Semaphorin-specific antibodies

ABSTRACT

A novel class of proteins, semaphorins, nucleic acids encoding semaphorins, semaphorin peptides, and methods of using semaphorins and semaphorin-encoding nucleic acids are disclosed. Semaphorin peptides and receptor agonists and antagonists provide potent modulators of nerve cell growth and regeneration. The invention provides pharmaceutical compositions, methods for screening chemical libraries for regulators of cell growth/differentiation; semaphorin gene-derived nucleic acids for use in genetic mapping, as probes for related genes, and as diagnostic reagents for genetic neurological disease; specific cellular and animal systems for the development of neurological disease therapy.

This application is a continuation of U.S. application Ser. No. 08/835,268, filed Apr. 8, 1997, now U.S. Pat. No. 5,807,826, which is a division of U.S. application Ser. No. 08/121,713, filed Sep. 13, 1993, now U.S. Pat. No. 5,639,856.

The research carried out in the subject application was supported in part by grants from the National Institutes of Health. The government may have rights in any patent issuing on this application.

TECHNICAL FIELD

The technical field of this invention concerns peptides, polypeptides, and polynucleotides involved in nerve cell growth.

BACKGROUND

The specificity of the wiring of the nervous system—the complex pattern of specific synaptic connections—begins to unfold during development as the growing tips of neurons—the growth cones—traverse long distances to find their correct targets. Along their journey, they are confronted by and correctly navigate a series of choice points in a remarkably unerring way to ultimately contact and recognize their correct target.

The identification of growth cone guidance cues is to a large extent, the holy grail of neurobiology. These are the compounds that tell neurons when to grow, where to grow, and when to stop growing. The medical applications of such compounds and their antagonists are enormous and include modulating neuronal growth regenerative capacity, treating neurodegenerative disease, and mapping (e.g. diagnosing) genetic neurological defects.

Over decades of concentrated research, various hypotheses of chemo-attractants and repellant, labeled pathways, cell adhesion molecules, etc. have been evoked to explain guidance. Recently, several recent lines of experiments suggest repulsion may play an important role in neuron guidance and two apparently unrelated factors (“Neurite Growth Inhibitor” and “Collapsin”) capable of inhibiting or collapsing growth cones have been reported.

RELEVANT LITERATURE

For a recent review of much of the literature in this field, see Goodman and Shatz (1993) Cell 72/Neuron 10, 77-98. A description of grasshopper fasciclin IV (now called G-Semaphorin I) appears in Kolodkin et al. (1992) Neuron 9, 831-845. Recent reports on Collapsin and Neurite Growth Inhibitor include Raper and Kapfhammer (1990) Neuron 4, 21-29, an abstract presented by Raper at the GIBCO-BRL Symposium on “Genes and Development/Function of Brain” on Jul. 26, 1993 and Schwab and Caroni (1988) J Neurosci 8, 2381 and Schnell and Schwab (1990) Nature 343, 269, respectively.

SUMMARY OF THE INVENTION

A novel class of proteins, semaphorins, nucleic acids encoding semaphorins, and methods of using semaphorins and semaphorin-encoding nucleic acids are disclosed. Semaphorins include the first known family of human proteins which function as growth cone inhibitors and a family of proteins involved in viral, particularly pox viral, pathogenesis and oncogenesis. Families of semaphorin-specific receptors, including receptors found on nerve growth cones and immune cells are also disclosed.

The invention provides agents, including semaphorin peptides, which specifically bind semaphorin receptors and agents, including semaphorin receptor peptides, which specifically bind semaphorins. These agents provide potent modulators of nerve cell growth, immune responsiveness and viral pathogenesis and find use in the treatment and diagnosis of neurological disease and neuro-regeneration, immune modulation including hypersensitivity and graft-rejection, and diagnosis and treatment of viral and oncological infection/diseases.

Semaphorins, semaphorin receptors, semaphorin-encoding nucleic acids, and unique portions thereof also find use variously in screening chemical libraries for regulators of semaphorin or semaphorin receptor-mediated cell activity, in genetic mapping, as probes for related genes, as diagnostic reagents for genetic neurological, immunological and oncological disease and in the production of specific cellular and animal systems for the development of neurological, immunological, oncological and viral disease therapy.

DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention discloses novel families of proteins important in nerve and immune cell function: the semaphorins and the semaphorin receptors. The invention provides agents, including semaphorin peptides, which specifically bind semaphorin receptors and agents, including semaphorin receptor peptides, which specifically bind semaphorins. These agents find a wide variety of clinical, therapeutic and research uses, especially agents which modulate nerve and/or immune cell function by specifically mimicing or interfering with semaphorin-receptor binding. For example, selected semaphorin peptides shown to act as semaphorin receptor antagonists are effective by competitively inhibiting native semaphorin association with cellular receptors. Thus, depending on the targeted receptor, these agents can be used to block semaphorin mediated neural cell growth cone repulsion or contact inhibition. Such agents find broad clinical application where nerve cell growth is indicated, e.g. traumatic injury to nerve cells, neurodegenerative disease, etc. A wide variety of semaphorin- and semaphorin receptor-specific binding agents and methods for identifying, making and using the same are described below.

Binding agents of particular interest are semaphorin peptides which specifically bind and antagonize a semaphorin receptor and semaphorin receptor peptides which specifically bind a semaphorin and prevent binding to a native receptor. While exemplified primarily with semaphorin peptides, much of the following description applies analogously to semaphorin receptor peptides.

The semaphorin peptides of the invention comprise a unique portion of a semaphorin and have semaphorin binding specificity. A “unique portion” of a semaphorin has an amino acid sequence unique to that disclosed in that it is not found in any previously known protein. Thus a unique portion has an amino acid sequence length at least long enough to define a novel peptide. Unique semaphorin portions are found to vary from about 5 to about 25 residues, preferably from 5 to 10 residues in length, depending on the particular amino acid sequence. Unique semaphorin portions are readily identified by comparing the subject semaphorin portion sequences with known peptide/protein sequence data bases. Preferred unique portions derive from the semaphorin domains (which exclude the Ig-like, intracellular and transmembrane domains as well as the signal sequences) of the disclosed semaphorin sequences, especially regions that bind the semaphorin receptor, especially that of the human varieties. Preferred semaphorin receptor unique portions derive from the semaphorin binding domains, especially regions with residues which contact the semaphorin ligand, especially that of the human varieties. Particular preferred peptides are further described herein.

The subject peptides may be free or coupled to other atoms or molecules. Frequently the peptides are present as a portion of a larger polypeptide comprising the subject peptide where the remainder of the polypeptide need not be semaphorin- or semaphorin receptor-derived. Alternatively, the subject peptide may be present as. a portion of a “substantially full-length” semaphorin domain or semaphorin receptor sequence which comprises or encodes at least about 200, preferably at least about 250, more preferably at least about 300 amino acids of a disclosed semaphorin/receptor sequence. Thus the invention also provides polypeptides comprising a sequence substantially similar to that of a substantially full-length semaphorin domain or a semaphorin receptor. “Substantially similar” sequences share at least about 40%, more preferably at least about 60%, and most preferably at least about 80% sequence identity. Where the sequences diverge, the differences are generally point insertions/deletions or conservative substitutions, i.e. a cysteine/threonine or serine substitution, an acidic/acidic or hydrophobic/hydrophobic amino acid substitution, etc.

The subject semaphorin peptides/polypeptides are “isolated”, meaning unaccompanied by at least some of the material with which they are associated in their natural state. Generally, an isolated peptide/polypeptide constitutes at least about 1%, preferably at least about 10%, and more preferably at least about 50% by weight of the total peptide/protein in a given sample. By pure peptide/polypeptide is intended at least about 90%, preferably at least 95%, and more preferably at least about 99% by weight of total peptide/protein. Included in the subject peptide/polypeptide weight are any atoms, molecules, groups, or polymers covalently coupled to the subject semaphorin/receptor peptide/polypeptide, especially peptides, proteins, detectable labels, glycosylations, phosphorylations, etc.

The subject peptides/polypeptides may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample and to what, if anything, the peptide/polypeptide is covalently linked. Purification methods include electrophoretic, molecular, immunological and chromatographic techniques, especially affinity chromatography and RP-HPLC in the case peptides. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer-Verlag, New York (1982).

The subject peptides/polypeptides generally comprise naturally occurring amino acids but D-amino acids or amino acid mimetics coupled by peptide bonds or peptide bond mimetics may also be used. Amino acid mimetics are other than naturally occurring amino acids that conformationally mimic the amino acid for the purpose of the requisite semaphorin/receptor binding specificity. Suitable mimetics are known to those of ordinary skill in the art and include β-γ-δ amino and imino acids, cyclohexylalanine, adamantylacetic acid, etc., modifications of the amide nitrogen, the α-carbon, amide carbonyl, backbone modifications, etc. See, generally, Morgan and Gainor (1989) Ann. Repts. Med. Chem 24, 243-252; Spatola (1983) Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol VII (Weinstein) and Cho et. al (1993) Science 261, 1303-1305 for the synthesis and screening of oligocarbamates.

The subject semaphorin peptides/polypeptides have a “semaphorin binding specificity” meaning that the subject peptide/polypeptide retains a molecular conformation specific to one or more of the disclosed semaphorins and specifically recognizable by a semaphorin-specific receptor, antibody, etc. As such, a semaphorin binding specificity may be provided by a semaphorin-specific immunological epitope, lectin binding site, etc., and preferably, a receptor binding site. Analogously, the semaphorin receptor peptides/polypeptides have a “semaphorin receptor binding specificity” meaning that these peptides/polypeptides retain a molecular conformation specific to one or more of the disclosed semaphorin receptors and specifically recognizable by a semaphorin, a receptor-specific antibody, etc.

“Specific binding” is empirically determined by contacting, for example a semaphorin-derived peptide with a mixture of components and identifying those components that preferentially bind the semaphorin. Specific binding is most conveniently shown by competition with labeled ligand using recombinant semaphorin peptide either in vitro or in cellular expression systems as disclosed herein. Generally, specific binding of the subject semaphorin has binding affinity of 10⁻⁶M, preferably 10⁻⁸M, more preferably 10⁻¹⁰M, under in vitro conditions as exemplified below.

The peptides/polypeptides may be modified or joined to other compounds using physical, chemical, and molecular techniques disclosed or cited herein or otherwise known to those skilled in the relevant art to affect their semaphorin binding specificity or other properties such as solubility, membrane transportability, stability, binding specificity and affinity, chemical reactivity, toxicity, bioavailability, localization, detectability, in vivo half-life, etc. as assayed by methods disclosed herein or otherwise known to those of ordinary skill in the art. For example, point mutations are introduced by site directed mutagenesis of nucleotides in the DNA encoding the disclosed semaphorin polypeptides or in the course of in vitro peptide synthesis.

Other modifications to further modulate binding specificity/affinity include chemical/enzymatic intervention (e.g. fatty acid-acylation, proteolysis, glycosylation) and especially where the peptide/polypeptide is integrated into a larger polypeptide, selection of a particular expression host, etc. In particular, many of the disclosed semaphorin peptides contain serine and threonine residues which are phosphorylated or dephosphorylated. See e.g. methods disclosed in Roberts et al. (1991) Science 253, 1022-1026 and in Wegner et al. (1992) Science 256, 370-373. Amino and/or carboxyl termini may be functionalized e.g., for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like. Many of the disclosed semaphorin peptides/polypeptides also contain glycosylation sites and patterns which may disrupted or modified, e.g. by enzymes like glycosidases or used to purify/identify the receptor, e.g. with lectins. For instance, N or O-linked glycosylation sites of the disclosed semaphorin peptides may be deleted or substituted for by another basic amino acid such as Lys or His for N-linked glycosylation alterations, or deletions or polar substitutions are introduced at Ser and Thr residues for modulating O-linked glycosylation. Glycosylation variants are also produced by selecting appropriate host cells, e.g. yeast, insect, or various mammalian cells, or by in vitro methods such as neuraminidase digestion. Useful expression systems include COS-7, 293, BHK, CHO, TM4, CV1, VERO-76, HELA, MDCK, BRL 3A, W138, Hep G2, MMT 060562, TRI cells, baculovirus systems, for examples. Other covalent modifications of the disclosed semaphorin peptides/polypeptides may be introduced by reacting the targeted amino acid residues with an organic derivatizing (e.g. methyl-3-[(p-azido-phenyl)dithio] propioimidate) or crosslinking agent (e.g. 1,1-bis(diazoacetyl)-2-phenylethane) capable of reacting with selected side chains or termini. For therapeutic and diagnostic localization, semaphorins and peptides thereof may be labeled directly (radioisotopes, fluorescers, etc.) or indirectly with an agent capable of providing a detectable signal, for example, a heart muscle kinase labeling site.

The following are 14 classes of preferred semaphorin peptides where bracketed positions may be occupied by any one of the residues contained in the brackets and “Xaa” signifies that the position may be occupied by any one of the 20 naturally encoded amino acids. These enumerated peptides maintain highly conserved structures which provide important semaphorin binding specificities;

(a)

[AspGlu]Cys[GlnLysArgAlaAsn]Asn[TyrPheVal]Ile (SEQ ID NO:1)

Cys[GlnLysArgAlaAsn]Asn[TyrPheVal]Ile[ArgLysGlnThr] (SEQ ID NO:2)

(b)

CysGlyThr[AsnGly][AlaSerAsn][TyrPheHisGly][LysArgHisAsnGln] (SEQ ID NO:3)

CysGlyThr[AsnGly][AlaSerAsn]XaaXaaPro (SEQ ID NO:4)

CysGlyThr[AsnGly]XaaXaaXaaProXaa[CysAsp] (SEQ ID NO:5)

CysGlyThrXaaXaaXaaXaaProXaa[CysAsp]XaaXaa[TyrIle] (SEQ ID NO:6)

(c)

[ArgIleGlnVal][GlyAla][LeuValLys][CysSer]Pro[PheTyr][AspAsn] (SEQ ID NO:7)

[CysSer]Pro[PheTyr][AspAsn]Pro[AspGluArgLys][HisLeuAsp] (SEQ ID NO:8)

GlyXaa[GlyAla]Xaa[CysSer]ProTyr[AspAsn]Pro (SEQ ID NO:9)

(d)

Leu[PheTyr]Ser[GlyAla]Thr[ValAsnAla]Ala (SEQ ID NO:10)

Leu[PheTyr]SerXaaThrXaaAla[AspGlu][PheTyr] (SEQ ID NO:11)

[PheTyr]Ser[GlyAla]Thr[ValAsnAla]Ala[AspGlu][PheTyr] (SEQ ID NO:12)

(e)

Leu[AsnAsp][AlaLys]ProAsnPheVal (SEQ ID NO:13)

(f)

PhePhePheArgGlu (SEQ ID NO:14)

PhePhe[PheTyr]ArgGlu[ThrAsn] (SEQ ID NO:15)

PhePheArgGlu[ThrAsn]Ala (SEQ ID NO:16)

Phe[PheTyr]ArgGlup[ThrAsn]Ala (SEQ ID NO:17)

TyrPhePhe[PheTyr]ArgGlu (SEQ ID NO:18)

[PheTyr]PhePhe[PheTyr]ArgGlu (SEQ ID NO:19)

[PheTyr][PheTyr][PheTyr]ArgGlu[ThrAsn]Ala (SEQ ID NO:20)

[IleVal][PheTyr]Phe[PheTyr][PheTyr]ArgGlu (SEQ ID NO:21)

Asp[LysPheTyr]Val[PheTyr][PheTyrIleLeu][PheTyrIleLeu][PheTyr] (SEQ ID NO:22)

[ValIle][PheTyr][PheTyrIleLeu][PheTyrIleLeu]Phe[ArgThr]Xaa[ThrAsn](SEQID NO:23)

[ValIle][PheTyr][PheTyrIleLeu][PheTyrIleLeu][PheTyr][ArgThr][GluAspVal][ThrAsn] (SEQ ID NO:24)

(g)

Glu[PheTyr]IleAsn[CysSer]GlyLys (SEQ ID NO:25)

[PheTyr]IleAsnCysGlyLys[AlaValIle] (SEQ ID NO:26)

(h)

Arg[ValIle][AlaGly][ArgGln][ValIle]CysLys (SEQ ID NO:27)

Arg[ValIle]Xaa[ArgGln][ValIle]CysXaaXaaAsp (SEQ ID NO:28)

GlyLys[ValAlaIle]XaaXaaXaaArg[ValAlaIle]XaaXaaXaaCysLys (SEQ ID NO:29)

(i)

[ArgLysAsn]Trp[ThrAlaSer][ThrAlaSer][PheTyrLeu]Leu[LysArg] (SEQ ID NO:30)

[PheTyr]Leu[LysArg][AlaSer]ArgLeu[AsnIle]Cys (SEQ ID NO:31)

[AsnIle]CysSer[[IleVal][ProSer]Gly (SEQ ID NO:32)

Trp[ThrAlaSer][ThrAlaSer][PheTyrLeu]LeuLys[AlaSerValIleLeu]XaaLeu (SEQ ID NO:33)

Trp[ThrAlaSer][ThrAlaSer]XaaLeuLysXaaXaaLeuXaaCys (SEQ ID NO:34)

TrpXaa[ThrSer]XaaLeuLysXaaXaaLeuXaaCys (SEQ ID NO:35)

(j)

[PheTyr][PheTyr][AsnAsp]GluIleGlnSer (SEQ ID NO:36)

[PheTyr]Pro[PheTyr][PheTyr][PheTyr][AsnAsp]Glu (SEQ ID NO:37)

(k)

GlySerAla[ValIleLeu]CysXaa[PheTyr] (SEQ ID NO:38)

SerAla[ValIleLeu]CysXaa[PheTyr]XaaMet (SEQ ID NO:39)

(l)

AsnSer[AsnAla]TrpLeu[ProAla]Val (SEQ ID NO:40)

(m)

[ValLeuIle]Pro[GluAspTyrSerPhe]ProArgProGly (SEQ ID NO:41)

[ValLeuIle]ProXaaPro[ArgAla]ProGlyXaaCys (SEQ ID NO:42)

Pro[GluAspTyrSerPhe]ProArgProGly[ThrGlnSer]Cys (SEQ ID NO:43)

(n)

AspPro[HisPheTyr]Cys[AlaGly]Trp (SEQ ID NO:44)

Pro[HisPheTyr]Cys[AlaGly]TrpAsp (SEQ ID NO:45)

AspProXaaCys[AlaGly]TrpAsp (SEQ ID NO:46)

CysXaaXaaXaaXaaAspProXaaCysTrpAsp (SEQ ID NO:47)

CysXaaXaaXaaAspProXaaCysXaaTrpAsp (SEQ ID NO:48)

CysXaaXaaAspProXaaCysXaaTrpAsp (SEQ ID NO:49)

CysXaaXaaCysXaaXaaXaaXaaAspXaaXaaCysXaaTrpAsp (SEQ ID NO:50)

CysXaaXaaCysXaaXaaXaaAspXaaXaaCysXaaTrpAsp (SEQ ID NO:51)

CysXaaXaaCysXaaXaaAspXaaXaaCysXaaTrpAsp (SEQ ID NO:52)

The following peptides represent particularly preferred members of each class:

(a)

AspCysGlnAsnTyrIle (SEQ ID NO:67)

(b)

CysGlyThr[AsnGly][AlaSer]XaaXaaPro (SEQ ID NO:68)

(c)

GlyXaa[SerCys]ProTyrAspPro (SEQ D NO:69)

(d)

LeuTyrSerGlyThr[ValAsnAla]Ala (SEQ D NO:70)

(e)

LeuAsnAlaProAsnPheVal (SEQ ID NO:71)

(f)

[PheTyr]PhePhe[PheTyr]ArgGlu (SEQ ID NO:19)

(g)

Glu[PheTyr]IleAsn[CysSer]GlyLys (SEQ ID NO:25)

(h)

Arg[ValIle]AlaArgValCysLys (SEQ ID NO:72)

(i)

Trp[ThrAla][ThrSer][PheTyr]LeuLys[AlaSer]ArgLu (SEQ ID NO:73)

(j)

ProPheTyrPhe[AsnAsp]GluIleGlnSer (SEQ ID NO:74)

(k)

GlySerAlaValCysXaa[PheTyr] (SEQ ID NO:75)

(l)

AsnSerAsnTrpLeu[ProAla]Val (SEQ ID NO:76)

(m)

Pro[GluAsp]ProArgProGly][ThrGlnSer]Cys (SEQ ID NO:77)

(n)

AspProTyrCys[AlaGly]TrpAsp (SEQ ID NO:78)

The following 14 classes are preferred peptides which exclude semaphorin peptides encoded in open reading frames of Variola major or Vaccinia viruses.

(a)

[AspGlu]Cys[GlnLysArgAlaAsn]Asn[TyrPheVal]Ile (SEQ ID NO:01)

Cys[GlnLysArgAlaAsn]Asn[TyrPheVal]Ile[ArgLysGlnThr] (SEQ ID NO:02)

(b)

CysGlyThr[AsnGly][AlaSer][TryrPheHisGly][LysArgHisAsnGln] (SEQ ID NO:79)

CysGlyThr[AsnGly][AlaSerAsn][TyrPheHis][LysArgHisAsnGln] (SEQ ID NO:80)

CysGlyThr[AsnGly][AlaSer]XaaXaaPro (SEQ ID NO:81)

(c)

[ArgIleGlnVal][GlyAla][LeuValLys][CysSer]Pro[PheTyr][AspAsn] (SEQ ID NO:07)

[CysSer]Pro[PheTYr][AspAsn]Pro[AspGluArgLys][HisLeuAsp] (SEQ ID NO:08)

GlyXaa[GlyAla]Xaa[CysSer]ProTyr[AspAsn]Pro (SEQ ID NO:09)

(d)

Leu[PheTyr]Ser[GlyAla]Thr[ValAsnAla]Ala (SEQ ID NO:10)

Leu[PheTyr]SerXaaThrXaaAla[AspGlu][PheTyr] (SEQ ID NO:11)

[PheTyr]Ser[GlyAla]Thr[ValAsnAla]Ala[AspGlu][PheTyr] (SEQ ID NO:12)

(e)

Leu[AsnAsp][AlaLys]ProAsnPheVal (SEQ ID NO:13)

(f)

PhePhePheArgGlu (SEQ ID NO:14)

PhePhe[PheTyr]ArgGlu[ThrAsn] (SEQ ID NO:15)

PhePheArgGlu[ThrAsn]Ala (SEQ ID NO:16)

Phe[PheTyr]ArgGlu[ThrAsn]Ala (SEQ ID NO:17)

TyrPhePhe[PheTyr]ArgGlu (SEQ ID NO:18)

[PheTyr]PhePhe[PheTyr]ArgGlu (SEQ ID NO:19)

[PheTyr][PheTyr][PheTyr]ArgGlu[ThrAsn]Ala (SEQ ID NO:20)

[IleVal][PheTyr]Phe[PheTyr][PheTyr]ArgGlu (SEQ ID NO:21)

Asp[LysPheTyr]Val[PheTyr][PheTyrLeu][PheTyrIleLeu][PheTyr] (SEQ ID NO:22)

Asp[LysPheTyr]Val[PheTyr][PheTyrIleLeu][PheTyrIle][PheTyr] (SEQ ID NO:82)

[ValIle][PheTyr][PheTyrLeu][PheTyrIleLeu]Phe[ArgThr]Xaa[ThrAsn] (SEQ ID NO:83)

[ValIle][PheTyr][PheTyrIleLeu][PheTyrIle]Phe[ArgThr]Xaa[ThrAsn] (SEQ ID NO:84)

[ValIle][PheTyr][PheTyrIleLeu][PheTyrIleLeu][PheArgXaa[ThrAsn] (SEQ ID NO:85)

[ValIle][PheTyr][PheTyrLeu][PheTyrIleLeu][PheTyr][ArgThr][GluAspVal][ThrAsn] (SEQ ID NO:86)

(g)

Glu[PheTyr]]IleAsn[CysSer]GlyLys (SEQ ID NO:25)

[PheTyr]IleAsnCysGlyLys[AlaValIle] (SEQ ID NO:26)

(h)

Arg[ValIle][AlaGly][ArgGln][ValIle]CysLys (SEQ ID NO:27)

Arg[ValIle]Xaa[ArgGln][ValIle]CySXaaXaaAsp (SEQ ID NO:28)

GlyLys[ValIle]XaaXaaXaaArg[ValAlaIle]XaaXaaXaaCysLys (SEQ ID NO:29)

(i)

[AraLysAsn]Trp[ThrAla][ThrAlaSer][PheTyrLeu]Leu[LysArg] (SEQ ID NO:87)

[PheTyr]Leu[LysArg][AlaSer]ArgLeu[AsnIle]Cys (SEQ ID NO:31)

[AsnIle]CysSer[IleVal][ProSer]Gly (SEQ ID NO:32)

Trp[ThrAla][ThrAlaSer][PheTyreLeu]LeuLys[AlaSerValIleLeu]XaaLeu (SEQ ID NO:88)

Trp[ThrAlaSer][ThrAlaSer][PheTyrLeu]LeuLys[AlaSerIleLeu]XaaLeu (SEQ ID NO:89)

Trp[ThrAla][ThrAlaSer]XaaLeuLysXaaXaaLeuXaaCys (SEQ ID NO:90)

(j)

[PheTyr][PheTyr][AsnAsp]GluIleGlnSer (SEQ ID NO:36)

[PheThr]Pro[PheTyr][PheTyr][PheTyr][AsnAsp]Glu (SEQ ID NO:37)

(k)

GlySerAla[ValIleLeu]CysXaa[PheTyr] (SEQ ID NO:38)

SerAla[ValIle]CysXaa[PheTyr]XaaMet (SEQ ID NO:39)

(l)

AsnSer[AsnAla]TrpLeu[ProAla]Val (SEQ ID NO:40)

(m)

[ValLeuIle]Pro[GluAspTyrSerPhe]ProArgProGly (SEQ ID NO:41)

[ValLeuIle]ProXaaProArgProGlyXaaCys (SEQ ID NO:91)

Pro[GluAspTyrSerPhe]ProArgProGly[ThrGlnSer]Cys (SEQ ED NO:43)

(n)

AspPro[HisPheTyr]Cys[AlaGly]Trp (SEQ ID NO:44)

Pro[HisPheTyr]Cys[AlaGly]TrpAsp (SEQ ID NO:45)

AspProXaaCys[AlaGly]TrpAsp (SEQ ED NO:46)

CysXaaXaaXaaXaaAspProXaaCysXaaTrpAsp (SEQ ID NO:47)

CysXaaXaaXaaAspProXaaCysXaaTrpAsp (SEQ ID NO:48)

CysXaaXaaAspProXaaCysXaaTrpAsp (SEQ ID NO:49)

CysXaaXaaCysXaaXaaXaaXaaAspXaaXaaCysXaaTrpAsp (SEQ ID NO:50)

CysXaaXaaCysXaaXaaXaaAspXaaXaaCysXaaTrpAsp (SEQ ED NO:51)

CysXaaXaaCysXaaXaaAspXaaXaaCysXaaTrpAsp (SEQ ID NO:52)

The following 2 class are prepared peptides which exclude semaphorin peptides encoded in open reading frames of Variola major or Vaccinia viruses Grasshopper Semaphorin I.

(f)

TyrPhePhe[PheTyr]ArgGlu (SEQ ID NO:18)

Asp[LysTyr]Val[PheTyr][PheTyrLeu][PheTyrIleLeu][PheTyr] (SEQ ID NO:92)

Asp[LysTyr]Val[PheTyr][PheTyrIleLeu][PheTyrIle][PheTyr] (SEQ ID NO:93)

[ValIle]Tyr[PheTyrLeu][PheTyrIleLeu]Phe[ArgThr]Xaa[ThrAsn] (SEQ ID NO:94)

[ValIle]Tyr[PheTyrIleLeu][PheTyrIle]Phe[ArgThr]Xaa[ThrAsn] (SEQ ID NO:95)

[ValIle]Tyr[PheTyrIleLeu][PheTyrIleLeu]PheArgXaa[ThrAsn] (SEQ ID NO:96)

Val[PheTyr][PheTyrLeu][PheTyrIleLeu][PheTyr][ArgThr][GluAspVal][ThrAsn](SEQID NO:97)

Val[PheTyr][PheTyrIleLeu][PheTyrIle][PheTyr][ArgThr][GluAspVal][ThrAsn](SEQID NO:98)

Val[PheTyr][PheTyrIleLeu][PheTyrIleLeu][PheTyr]Arg[GluAspVal][ThrAsn](SEQID NO:99)

(n)

CysXaaXaaXaaAspProXaaCysXaaTrpAsp (SEQ ID NO:48)

CysXaaXaaAspProXaaCysXaaTrpAsp (SEQ ID NO:49)

CysXaaXaaCysXaaXaaXaaAspXaaXaaCysXaaTrpAsp (SEQ ID NO:51)

CysXaaXaaCysXaaXaaAspXaaXaaCysXaaTrpAsp (SEQ ID NO:52)

The following 5 classes include peptides which encompass peptides encoded in open reading frames of Variola major or Vacacinia viruses. Accordingly, in the event that these viral peptides are not novel per se, the present invention discloses a hitherto unforseen and unforseeable utility for these peptides as immunosuppressants and targets of anti-viral therapy.

(b)

CysGlyThr[AsnGly][AlaSerAsn][TyrPheHisGly][LysArgHisAsnGln] (SEQ ID NO:03)

CysGlyThr[AsnGly][AlaSerAsn]XaaXaaPro (SEQ ID NO:04)

CysGlyThr[AsnGly]XaaXaaXaProXaa[CysAsp] (SEQ ID NO:05)

CysGlyThrXaaXaaXaaXaaProXaa[CysAsp]XaaXaa[TyrIle] (SEQ ID NO:06)

(f)

Asp[LysPheTyr]Val[PheTyr][PheTyrIleLeu][PheTyrIleLeu][PheTyr] (SEQ ID NO:22)

[ValIle][PheTyr][PheTyrIleLeu][PheTyrIleLeu]Phe[ArgThr]Xaa[ThrAsn] (SEQ ID NO:23)

Val[PheTyr][PheTyrIleLeu][PheTyrIleLeu][PheTyr][ArgThr][GluVal][ThrAsn] (SEQ ID NO:100)

(i)

[ArgLysAsn]Trp[ThrAlaSer][ThrAlaSer][PheTyrLeu]Leu[LysArg] (SEQ ID NO:30)

Trp[ThrAlaSer][PheTyrLeu]LeuLys[AlaSerValIleLeu]XaaLeu (SEQ ID NO:33)

Trp[ThrAlaSer][ThrAlaSer]XaaLeuLysXaaXaaLeuXaaCys (SEQ ID NO:34)

TrpXaa[ThrSer]XaaLeuLysXaaXaaLeuXaaCys (SEQ ID NO:35)

(k)

SerAla[ValIleLeu]CysXaa[PheTyr]XaaMet (SEQ IID NO:39)

(m)

[ValLeuIle]ProXaaPro[ArgAla]ProGlyXaaCys (SEQ D NO:42)

The disclosed semaphorin sequence data are used to define a wide variety of other semaphorin- and semaphorin receptor-speicifc binding agents using immunologic, chromatographic or synthetic methods available to those skilled in the art.

Of particular significance are peptides comprising unique portions of semaphorin-specific receptors and polypeptides comprising a sequence substantially similar to that of a substantially full-length semaphorin receptor. Using semaphorin peptides, these receptors are identified by a variety of techniques known to those skilled in the art where a ligand to the target receptor is known, including expression cloning as set out in the exemplification below. For other examples of receptor isolaton with known ligand using expression cloning, see, Staunton et al (1989) Nature 339, 61; Davis et al (1991) Science 253, 59; Lin et al (1992) Cell 68, 775; Gearing et al (1989) EMBO 8, 3667; Aruffo and Seed (1987) PNAS 84, 8573 and refrees therein. Generally, COS cells are transfected to express a cDNA mary or PCR product and cells producing peptides/polypeptides which bind a semaphorin/receptor peptide/polypeptide are isolated. For neurosemaphorin receptors, fetal brain cDNA libraries are preferred; for immunosemaphorin receptors, libraries derived from activated lymphoid or myeloid cell lines or tissue derived from sites of inflammation or delayed-type hypersensitivity are preferred; and for semaphorin and semaphorin receptor variants used by tumor cells to evade immune surveillance or suppress an immune response (oncosemaphorins), libraries derived from cancerous tissue or tumor cell lines resistant to the host immune system are preferred. Alternatively, PCR primers based upon known semaphorin/receptor sequences such as those disclosed herein are used to amplify PCR product from such tissues/cells. Other receptor/ligand isolation methods using immobilized ligand or antibody are known to those skilled in the art.

Semaphorin receptor peptides with receptor binding specificity are identified by a variety of ways including having conserved consensus sequences with other semaphorin receptors, by crosslinking to ligand or receptor-specific antibody, or preferably, by screening such peptides for semaphorin binding or disruption of semaphorin-receptor binding. Methods for identifying semaphorin receptor peptides with the requisite binding activity are described herein or otherwise known to those skilled in the art. By analogous methods, semaphorin receptor peptides are used to define additional semaphorin peptides with semaphorin binding specificity, particularly receptor specificity.

The various semaphorin and semaphorin receptor peptides are used to define functional domains of semaphorins, identify compounds that associate with semaphorins, design compounds capable of modulating semaphorin-mediated nerve and immune cell function, and define additional semaphorin and semaphorin receptor-specific binding agents. For example, semaphorin mutants, including deletion mutants are generated from the disclosed semaphorin sequences and used to identify regions important for specific protein-ligand or protein-protein interactions, for example, by assaying for the ability to mediate repulsion or preclude aggregation in cell-based assays as described herein. Further, x-ray crystallographic data of the disclosed protein are used to rationally design binding molecules of determined structure or complementarity for modulating growth cone growth and guidance.

Additional semaphorin- and receptor-specific agents include specific antibodies that can be modified to a monovalent form, such as Fab, Fab′, or Fv, specifically binding oligopeptides or oligonucleotides and most preferably, small molecular weight organic receptor antagonists. For example, the disclosed semaphorin and receptor peptides are used as immunogens to generate semaphorin- and receptor-specific polyclonal or monoclonal antibodies. See, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, for general methods. Anti-idiotypic antibody, especially internal imaging anti-ids are also prepared using the disclosures herein.

In addition to semaphorin and semaphorin-receptor derived polypeptides and peptides, other prospective agents are screened from large libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means. See, e.g. Houghten et al. and Lam et al (1991) Nature 354, 84 and 81, respectively and Blake and Litzi-Davis (1992), Bioconjugate Chem 3, 510.

Useful agents are identified with a range of assays employing a compound comprising the subject peptides or encoding nucleic acids. A wide variety of in vitro, cell-free binding assays, especially assays for specific binding to immobilized compounds comprising semaphorin or semaphorin receptor peptide find convenient use. While less preferred, cell-based assays may be used to determine specific effects of prospective agents on semaphorin-receptor binding may be assayed. Optionally, the intracellular C-terminal domain is substituted with a sequence encoding a oligopeptide or polypeptide domain that provides a detectable intracellular signal upon ligand binding different from the natural receptor. Useful intracellular domains include those of the human insulin receptor and the TCR, especially domains with kinase activity and domains capable of triggering calcium influx which is conveniently detected by fluorimetry by preloading the host cells with Fura-2. More preferred assays involve simple cell-free in vitro binding of candidate agents to immobilized semaphorin or receptor peptides, or vice versa. See, e.g. Fodor et al (1991) Science 251, 767 for light directed parallel synthesis method. Such assays are amenable to scale-up, high throughput usage suitable for volume drug screening.

Useful agents are typically those that bind to a semaphorin or disrupt the association of a semaphorin with its receptor. Preferred agents are semaphorin-specific and do not cross react with other neural or lymphoid cell membrane proteins. Useful agents may be found within numerous chemical classes, though typically they are organic compounds; preferably small organic compounds. Small organic compounds have a molecular weight of more than 150 yet less than about 4,500, preferably less than about 1500, more preferably, less than about 500. Exemplary classes include peptides, saccharides, steroids, heterocyclics, polycyclics, substituted aromatic compounds, and the like.

Selected agents may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents. For example, where peptide agents are identified, they may be modified in a variety of ways as described above, e.g. to enhance their proteolytic stability. Other methods of stabilization may include encapsulation, for example, in liposomes, etc.

The subject binding agents may be prepared in a variety of ways known to those skilled in the art. For example, peptides under about 60 amino acids can be readily synthesized today using conventional commercially available automatic synthesizers. Alternatively, DNA sequences may be prepared encoding the desired peptide and inserted into an appropriate expression vector for expression in a prokaryotic or eukaryotic host. A wide variety of expression vectors are available today and may be used in conventional ways for transformation of a competent host for expression and isolation. If desired, the open reading frame encoding the desired peptide may be joined to a signal sequence for secretion, so as to permit isolation from the culture medium. Methods for preparing the desired sequence, inserting the sequence into an expression vector, transforming a competent host, and growing the host in culture for production of the product may be found in U.S. Pat. Nos. 4,710,473, 4,711,843 and 4,713,339.

For therapeutic uses, the compositions and agents disclosed herein may be administered by any convenient way, preferably parenterally, conveniently in a pharmaceutically or physiologically acceptable carrier, e.g., phosphate buffered saline, saline, deionized water, or the like. Typically, the compositions are added to a retained physiological fluid such as blood or synovial fluid. For CNS administration, a variety of techniques are available for promoting transfer of the therapeutic across the blood brain barrier including disruption by surgery or injection, drugs which transciently open adhesion contact between CNS vasculature endothelial cells, and compounds which fascilitate translocation through such cells. As examples, many of the disclosed therapeutics are amenable to directly injected or infused, contained within implants e.g. osmotic pumps, grafts comprising appropriately transformed cells. Generally, the amount administered will be empirically determined, typically in the range of about 10 to 1000 μg/kg of the recipient. For peptide agents, the concentration will generally be in the range of about 50 to 500 μg/ml in the dose administered. Other additives may be included, such as stabilizers, bactericides, etc. These additives will be present in conventional amounts.

The invention provides isolated nucleic acid sequences encoding the disclosed semaphorin and semaphorin receptor peptides and polypeptides, including sequences substantially identical to sequences encoding such polypeptides. An “isolated” nucleic acid sequence is present as other than a naturally occurring chromosome or transcript in its natural state and typically is removed from at least some of the nucleotide sequences with which it is normally associated with on a natural chromosome. A complementary sequence hybridizes to a unique portion of the disclosed semaphorin sequence under low stringency conditions, for example, at 50° C. and SSC (0.9 M saline/0.09 M sodium citrate) and that remains bound when subject to washing at 55° C. with SSC. Regions of non-identity of complementary nucleic acids are preferably or in the case of homologous nucleic acids, a nucleotide change providing a redundant codon. A partially pure nucleotide sequence constitutes at least about 5%, preferably at least about 30%, and more preferably at least about 90% by weight of total nucleic acid present in a given fraction.

Unique portions of the disclosed nucleic acid sequence are of length sufficient to distinguish previously known nucleic acid sequences. Thus, a unique portion has a nucleotide sequence at least long enough to define a novel oligonucleotide. Preferred nucleic acid portions encode a unique semaphorin peptide. The nucleic acids of the invention and portions thereof, other than those used as PCR primers, are usually at least about 60 bp and usually less than about 60 kb in length. PCR primers are generally between about 15 and 100 nucleotides in length.

Nucleotide (cDNA) sequences encoding several full length semaphorins are disclosed in herein. The invention also provides for the disclosed sequences modified by transitions, transversions, deletions, insertions, or other modifications such as alternative splicing and also provides for genomic semaphorin sequences, and gene flanking sequences, including regulatory sequences; included are DNA and RNA sequences, sense and antisense. Preferred DNA sequence portions include portions encoding the preferred amino acid sequence portions disclosed above. For antisense applications where the inhibition of semaphorin expression is indicated, especially useful oligonucleotides are between about 10 and 30 nucleotides in length and include sequences surrounding the disclosed ATG start site, especially the oligonucleotides defined by the disclosed sequence beginning about 5 nucleotides before the start site and ending about 10 nucleotides after the disclosed start site. Other especially useful semaphorin mutants involve deletion or substitution modifications of the disclosed cytoplasmic C-termini of transmembrane semaphorins. Accordingly, semaphorin mutants with semaphorin binding affinities but with altered intracellular signal transduction capacities are produced.

For modified semaphorin-encoding sequences or related sequences encoding proteins with semaphorin-like functions, there will generally be substantial sequence identity between at least a segment thereof and a segment encoding at least a portion of the disclosed semaphorin sequence, preferably at least about 60%, more preferably at least 80%, most preferably at least 90% identity. Homologous segments are particularly within semaphorin domain-encoding regions and regions encoding protein domains involved in protein-protein, particularly semaphorin-receptor interactions and differences within such segments are particularly conservative substitutions. Typically, the invention's semaphorin peptide encoding polynucleotides are associated with heterologous sequences. Examples of such heterologous sequences include regulatory sequences such as promoters, enhancers, response elements, signal sequences, polyadenylation sequences, etc., introns, 5′ and 3′ noncoding regions, etc. Other useful heterologous sequences are known to those skilled in the art or otherwise disclosed references cited herein. According to a particular embodiment of the invention, portions of the semaphorin encoding sequence are spliced with heterologous sequences to produce soluble, secreted fusion proteins, using appropriate signal sequences and optionally, a fusion partner such as β-Gal.

The disclosed sequences are also used to identify and isolate other natural semaphorins and analogs. In particular, the disclosed nucleic acid sequences are used as hybridization probes under low-stringency or PCR primers, e.g. oligonucleotides encoding functional semaphorin domains are ³²P-labeled and used to screen λcDNA libraries at low stringency to identify similar cDNAs that encode proteins with related functional domains. Additionally, nucleic acids encoding at least a portion of the disclosed semaphorin are used to characterize tissue specific expression of semaphorin as well as changes of expression over time, particularly during organismal development or cellular differentiation.

The semaphorin encoding nucleic acids can be subject to alternative purification, synthesis, modification, sequencing, expression, transfection, administration or other use by methods disclosed in standard manuals such as Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor), Current Protocols in Molecular Biology (Eds. Aufubel, Brent, Kingston, More, Feidman, Smith and Stuhl, Greene Publ. Assoc., Wiley-Interscience, NY, N.Y., 1992) or that are otherwise known in the art. For example, the nucleic acids can be modified to alter stability, solubility, binding affinity and specificity, etc. semaphorin-encoding sequences can be selectively methylated, etc. The nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescers, biotinylation, etc.

The invention also provides vectors comprising nucleic acids encoding semaphorin. peptides, polypeptides or analogs. A large number of vectors, including plasmid and viral vectors, have been described for expression in a variety of eukaryotic and prokaryotic hosts. Advantageously, vectors may also include a promotor operably linked to the semaphorin-encoding portion. Vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance. The inserted semaphorin coding sequences may be synthesized, isolated from natural sources, prepared as hybrids, etc. Suitable host cells may be transformed/transfected/infected by any suitable method including electroporation, CaCl₂ mediated DNA uptake, viral infection, microinjection, microprojectile, or other methods.

Appropriate host cells include bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells. Of particular interest are E. coli, B. subtilis, Saccharomyces cerevisiae, SF9 cells, C129 cells, 293 cells, Neurospora, and CHO, COS, HeLa cells, immortalized mammalian myeloid and lymphoid cell lines, and pluripotent cells, especially mammalian ES cells and zygotes. Preferred replication systems include M13, ColE1, SV40, baculovirus, lambda, adenovirus, AAV, BPV, etc. A large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner of manipulation, etc. are known in the art. Under appropriate expression conditions, host cells can be used as a source of recombinantly produced semaphorins or analogs.

For the production of stably transformed cells and transgenic animals, nucleic acids encoding the disclosed semaphorins may be integrated into a host genome by recombination events. For example, such a sequence can be microinjected into a cell, and thereby effect homologous recombination at the site of an endogenous gene, an analog or pseudogene thereof, or a sequence with substantial identity to an semaphorin-encoding gene. Other recombination-based methods such as nonhomologous recombinations, deletion of endogenous gene by homologous recombination, especially in pluripotent cells, etc., provide additional applications. Preferred transgenics and stable transformants over-express the disclosed receptor gene and find use in drug development and as a disease model. Alternatively, knock-out cells and animals find use in development and functional studies. Methods for making transgenic animals, usually rodents, from ES cells or zygotes are known to those skilled in the art.

The compositions and methods disclosed herein may be used to effect gene therapy. See, e.g. Zhu et al. (1993) Science 261, 209-211; Gutierrez et al. (1992) Lancet 339, 715-721. For example, cells are transfected with semaphorin sequences operably linked to gene regulatory sequences capable of effecting altered semaphorin expression or regulation. To modulate semaphorin translation, cells may be transfected with complementary antisense polynucleotides. For gene therapy involving the transfusion of semaphorin transfected cells, administration will depend on a number of variables that are ascertained empirically. For example, the number of cells will vary depending on the stability of the transfused cells. Transfusion media is typically a buffered saline solution or other pharmacologically acceptable solution. Similarly the amount of other administered compositions, e.g. transfected nucleic acid, protein, etc., will depend on the manner of administration, purpose of the therapy, and the like.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

I. Isolation and Characterization of Grasshopper Semaphorin I (SEQ ID NOS:57 & 58) (Previously Referred to as Fasciclin IV)

In order to identify cell surface molecules that function in selective fasciculation, a series of monoclonal antibody (MAb) screens was conducted. The immunogen used for most of these screens was membranes from the longitudinal connectives (the collection of longitudinal axons) between adjacent segmental ganglia of the nervous system of the larval grasshopper. From these screens, MAb 3B11 and 8C6 were used to purify and characterize two surface glycoproteins, fasciclin I and fasciclin II, see, Bastiani et al., 1987; the genes encoding both were subsequently cloned, see, Snow et al. 1989, Zinn et al. 1988, and Harrelson and Goodman, 1988.

Another MAb isolated during these screens, MAb 6F8, was chosen for the present study because, just as with fasciclin I and fasciclin II, the antigen recognized by this MAb is expressed on a different but overlapping subset of axon pathways in the developing CNS. The 6F8 antigen appears to be localized on the outside of cell surfaces, as indicated by MAb binding when incubated both in live preparations, and in fixed preparations in which no detergents have been added. Because the 6F8 antigen is a surface glycoprotein expressed on a subset of axon fascicles (see below), we call it fasciclin IV.

Fasciclin IV expression begins early in embryonic development before axonogenesis. At 29% of development, expression is seen on the surface of the midline mesectodermal cells and around 5-7 neuroblasts and associated ectodermal cells per hemisegment. This expression is reminiscent of the mesectodermal and neuroblast-associated expression observed with both fasciclin I and fasciclin II; however, in each case, the pattern resolves into a different subset of neuroblasts and associated ectodermal cells.

At 32% of development, shortly after the onset of axonogenesis in the CNS, fasciclin IV expression is seen on the surface of the axons and cell bodies of the three pairs of MP4, MP5, and MP6 midline progeny, the three U motoneurons, and on several unidentified neurons in close proximity to the U's. This is in contrast to fasciclin II, which at this stage is expressed on the MP1 and dMP2 neurons, and fasciclin I, which is expressed on the U neurons but not on any midline precursor progeny.

The expression of fasciclin IV on a subset of axon pathways is best observed around 40% of development, after the establishment of the first longitudinal and commissural axon pathways . At this stage, the protein is expressed on two longitudinal axon fascicles, a subset of commissural axon fascicles, a tract extending anteriorly along the midline, and a subset of fascicles in the segmental nerve (SN) and intersegmental nerve (ISN) roots.

Specifically, fasciclin IV is expressed on the U fascicle, a longitudinal pathway (between adjacent segmental neuromeres) pioneered in part by the U neurons, and on the A/P longitudinal fascicle (in part an extension of the U fascicle within each segmental neuromere. In addition, fasciclin IV is also expressed on a second narrower, medial, and more ventral longitudinal pathway. The U axons turn and exit the CNS as they pioneer the ISN; the U's and many other axons within the ISN express fasciclin IV. The continuation of the U fascicle posterior to the ISN junction is also fasciclin IV-positive. The specificity of fasciclin IV for distinct subsets of longitudinal pathways can be seen by comparing fasciclin IV and fasciclin II expression in the same embryo; fasciclin IV is expressed on the U and A/P pathways whereas fasciclin II is expressed on the MP1 pathway.

The axons in the median fiber tract (MFT) also express fasciclin IV. The MFT is pioneered by the three pairs of progeny of the midline precursors MP4, MP5, and MP6. The MFT actually contains three separate fascicles. The axons of the two MP4 progeny pioneer the dorsal MFT fascicle and then bifurcate at the posterior end of the anterior commnissure; whereas the axons of the two MP6 progeny pioneer the ventral MFT fascicle and then bifurcate at the anterior end of the posterior commissure. Fasciclin IV is expressed on the cell bodies of the six MP4, MP5, and MP6 neurons, and on their growth cones and axons as they extend anteriorly in the MFT and bifurcate in one of the two commissures. However, this expression is regional in that once these axons bifurcate and begin to extend laterally across the longitudinal pathways and towards the peripheral nerve roots, their expression of fasciclin IV greatly decreases. Thus, fasciclin IV is a label for the axons in the MFT and their initial bifurcations in both the anterior and posterior commissures. It appears to be expressed on other commissural fascicles as well. However, the commissural expression of fasciclin IV is distinct from the transient expression of fasciclin II along the posterior edge of the posterior commissure, or the expression of fasciclin I on several different commissural axon fascicles in both the anterior and posterior commissure (Bastiani et al., 1987; Harrelson and Goodman, 1988).

Fasciclin IV is also expressed on a subset of motor axons exiting the CNS in the SN. The SN splits into two major branches, one anterior and the other posterior, as it exits the CNS. Two large bundles of motoneuron axons in the anterior branch express fasciclin IV at high levels; one narrow bundle of motoneuron axons in the posterior branch expresses the protein at much lower levels Fasciclin IV is also expressed on many of the axons in the ISN.

The CNS and nerve root expression patterns of fasciclin IV, fasciclin I, and fasciclin II at around 40% of embryonic development are summarized below. Although there is some overlap in their patterns (e.g., both fasciclin IV and fasciclin I label the U axons), these three surface glycoproteins label distinct subsets of axon pathways in the developing CNS.

Fasciclin IV is Expressed on Epithelial Bands in the Developing Limb Bud

Fasciclin IV is expressed on the developing limb bud epithelium in circumferential bands; at 34.5% of development these bands can be localized with respect to constrictions in the epithelium that mark presumptive segment boundaries. In addition to a band just distal to the trochanter/coxa segment boundary, bands are also found in the tibia, femur, coxa, and later in development a fifth band is found in the tarsus. Fasciclin IV is also expressed in the nascent chordotonal organ in the dorsal aspect of the femur. The bands in the tibia, trochanter, and coxa completely encircle the limb. However, the femoral band is incomplete, containing a gap on the anterior epithelia of this segment.

The position of the Ti1 axon pathway with respect to these bands of fasciclin IV-positive epithelia suggests a potential role for fasciclin IV in guiding the Ti1 growth cones. First, the band of fasciclin IV expression in the trochanter, which is approximately three epithelial cell diameters in width when encountered by the Ti1 growth cones, is the axial location where the growth cones reorient from proximal migration to circumferential branch extension. The Tr1 cell, which marks the location of the turn, lies within this band, usually over the central or the proximal cell tier. Secondly, although there is a more distal fasciclin IV expressing band in the femur, where a change in Ti1 growth is not observed, there exists a gap in this band such that fasciclin IV expressing cells are not traversed by the Ti1 growth cones. The Ti1 axons also may encounter a fasciclin IV expressing region within the coxa, where interactions between the growth cones, the epithelial cells, and the Cx1 guidepost cells have not yet been investigated.

In addition to its expression over the surface of bands of epithelial cells, fasciclin IV protein, as visualized with MAb 6F8, is also found on the basal surface of these cells in a punctate pattern. This punctate staining is not an artifact of the HRP immunocytochemistry since fluorescent visualization of MAb 6F8 is also punctate. The non-neuronal expression of fasciclin IV is not restricted to limb buds. Circumferential epithelial bands of fasciclin IV expression are also seen on subesophageal mandibular structures and on the developing antennae.

MAb Directed Against Fasciclin IV Can Alter the Formation of the Ti1 Axon Pathway in the Limb Bud

The expression of fasciclin IV on an epithelial band at a key choice point in the formation of the Ti1 axon pathway led us to ask whether this protein is involved in growth cone guidance at this location. To answer this question, we cultured embryos, or epithelial fillets (e. g., O'Connor et al., 1990), during the 5% of development necessary for normal pathway formation, either in the presence or absence of MAb 6F8 or 6F8 Fab fragments. Under the culture conditions used for these experiments, defective Ti1 pathways are observed in 14% of limbs (Chang et al., 1992); this defines the baseline of abnormalities observed using these conditions. For controls we used other MAbs and their Fab fragments that either bind to the surfaces of these neurons and epithelial cells (MAb 3B11 against the surface protein fasciclin I) or do not (MAb 4D9 against the nuclear protein engrailed; Patel et al., 1989). To assess the impact of MAb 6F8 on Ti1 pathway formation, we compared the percentage of aberrant pathways observed following treatment with MAb 6F8 to that observed with MAbs 3B11 and 4D9. Our cultures began at 32% of development when the Ti1 growth cones have not yet reached the epithelium just distal to the trochanter/coxa boundary and therefore have not encountered epithelial cells expressing fasciclin IV. Following approximately 30 hours in culture (˜4% of development), embryos were fixed and immunostained with antibodies to HRP in order to visualize the Ti1 axons and other neurons in the limb bud. Criteria for scoring the Ti1 pathway, and the definition of “aberrant”, are described in detail in the Experimental Procedures.

Although MAb 6F8 does not arrest pathway formation, several types of distinctive, abnormal pathways are observed. These defects generally begin where growth cones first contact the fasciclin IV expressing cells in the trochanter. Normally, the Ti1 neurons each have a single axon, and the axons of the two cells are fasciculated in that portion of the pathway within the trochanter. Following treatment with MAb 6F8, multiple long axon branches are observed within, and proximal to, the trochanter. Two major classes of pathways are taken by these branches; in 36% of aberrant limbs, multiple, long axon branches extend ventrally in the region distal to the Cx1 cells which contains the band of fasciclin IV expressing epithelial cells. In the ventral region of the trochanter, these branches often independently turn proximally to contact the Cx1 cells, and thus complete the pathway in this region.

In the second major class of pathway defect, seen in 47% of aberrant limbs, axon branches leave the trochanter at abnormal, dorsal locations, and extend proximally across the trochanter/coxa boundary. These axons then veer ventrally, often contacting the Cx1 neurons. The remaining 17% of defects include defasciculation distal to the trochanter, axon branches that fail to turn proximally in the ventral trochanter and continue into the posterior compartment of the limb, and axon branches which cross the trochanter/coxa boundary and continue to extend proximally without a ventral turn.

When cultured in the presence of MAb 6F8, 43% of limbs exhibited malformed Ti1 pathways (n=381) as compared to 11% with MAb 3B11 (n=230) and 5% with MAb 4D9 (n=20). These percentages are pooled from treatments with MAbs concentrated from hybridoma supernatant, IgGs isolated from these supernatants, and Fab fragments isolated from these IgG preparations (see Experimental Procedures). The frequency of malformed Ti1 pathways and the types of defects observed showed no significant variation regard less of the method of antibody preparation or type of antibody used. Since Fabs show similar results as IgGs, the effects of MAb 6F8 are not due to cross linking by the bivalent IgG.

In summary, following treatment with MAb 6F8, the Ti1 pathway typically exhibits abnormal morphology beginning just distal to the trochanter and at the site of fasciclin IV expression. The two most common types of Ti1 pathway defects described above occur in 36% of experimental limbs (treated with MAb 6F8), but are seen in only 4% of control limbs (treated with MAbs 3 B11 and 4D9).

Fasciclin IV cDNAs Encode a Novel Integral Membrane Protein

Grasshopper fasciclin IV was purified by passing crude embryonic grasshopper lysates over a MAb 6F8 column. After affinity purification, the protein was eluted, precipitated, denatured, modified at cysteines, and digested with either trypsin or Lys-C. Individual peptides were resolved by reverse phase HPLC and microsequenced using standard methods.

The amino acid sequences derived from these proteolytic fragments were used to generate oligonucleotide probes for PCR experiments, resulting in products that were used to isolate cDNA clones from the Zinn embryonic grasshopper cDNA library (Snow et al., 1988). Sequence analysis of these cDNAs reveals a single open reading frame (ORF) encoding a protein with two potential hydrophobic stretches of amino acids: an amino-terminal signal sequence of 20 residues and (beginning at amino acid 627) a potential transmembrane domain of 25 amino acids. Thus, the deduced protein has an extracellular domain of 605 amino acids, a transmembrane domain, and a cytoplasmic domain of 78 amino acids. The calculated molecular mass of the mature fasciclin IV protein is 80 kd and is confirmed by Western blot analysis of the affinity purified and endogenous protein as described below. The extracellular domain of the protein includes 16 cysteine residues that fall into three loose clusters but do not constitute a repeated domain and are not similar to other known motifs with cysteine repeats. There are also six potential sites for N-linked glycosylation in the extracellular domain. Treatment of affinity purified fasciclin IV with N-Glycanase demonstrates that fasciclin IV does indeed contain N-linked oligosaccharides. Fasciclin IV shows no sequence similarity when compared with other proteins in the PIR data base using BLASTP (Altschul et al., 1990), and is therefore a novel type I integral membrane protein.

A polyclonal antiserum directed against the cytoplasmic domain of the protein encoded by the fasciclin IV cDNA was used to stain grasshopper embryos at 40% of development. The observed staining pattern was identical to that seen with MAb 6F8. On Western blots, this antiserum recognizes the protein we affinity purified using MAb 6F8 and then subjected to microsequence analysis. Additionally, the polyclonal serum recognizes a protein of similar molecular mass from grasshopper embryonic membranes. Taken together these data indicate that the sequence we have obtained is indeed fasciclin IV.

Four other cell surface proteins that label subsets of axon pathways in the insect nervous system (fasciclin I, fasciclin II, fasciclin III, and neuroglian) are capable of mediating homophilic cell adhesion when transfected into S2 cells in vitro (Snow et al., 1989; Elkins et al., 1990b; Grenningloh et al., 1990). To ask whether fasciclin IV can function as a homophilic cell adhesion molecule, the fasciclin IV cDNA with the complete ORF was placed under the control of the inducible metallothionein promoter (Bunch et al., 1988), transfected into S2 cells, and assayed for its ability to promote adhesion in normally non-adhesive S2 cells. Following induction with copper, fasciclin IV was synthesized in these S2 cells as shown by Western blot analysis and cell surface staining of induced S2 cells with the polyclonal antiserum described above.

We observed no evidence for aggregation upon induction of fasciclin IV expression, thus suggesting that, in contrast to the other four proteins, fasciclin IV does not function as a homophilic cell adhesion molecule. Alternatively, fasciclin IV-mediated aggregation might require some further posttranslational modification, or co-factor, not supplied by the S2 cells, but clearly this protein acts differently in the S2 cell assay than the other four axonal glycoproteins previously tested. This is consistent with the pattern of fasciclin IV expression in the embryonic limb since only the epithelial cells and not the Ti1 growth cones express fasciclin IV, and yet antibody blocking experiments indicate that fasciclin IV functions in the epithelial guidance of these growth cones. Such results suggest that fasciclin IV functions in a heterophilic adhesion or signaling system.

DISCUSSION

Fasciclin IV is expressed on groups of axons that fasciculate in the CNS, suggesting that, much like other insect axonal glycoproteins, it functions as a homophilic cell adhesion molecule binding these axons together. Yet, in the limb bud, fasciclin IV is expressed on a band of epithelium but not on the growth cones that reorient along this band, suggesting a heterophilic function. That fasciclin IV functions in a heterophilic rather than homophilic fashion is supported by the lack of homophilic adhesion in S2 cell aggregation assays. In contrast, fasciclin I, fasciclin II, fasciclin III, and neuroglian all can function as homophilic cell adhesion molecules (Snow et al., 1989; Elkins et al., 1990b; Grenningloh et al., 1990).

cDNA sequence analysis indicates that fasciclin IV is an integral membrane protein with a novel sequence not related to any protein in the present data base. Thus, fasciclin IV represents a new type of protein that functions in the epithelial guidance of pioneer growth cones in the developing limb bud. Given its expression on a subset of axon pathways in the developing CNS, fasciclin IV functions in the guidance of CNS growth cones as well.

The results from the MAb blocking experiments illuminate several issues in Ti1 growth cone guidance and axon morphogenesis in the limb. First, the most striking change in growth cone behavior in the limb is the cessation of proximal growth and initiation of circumferential extension of processes upon encountering the trochanter/coxa boundary region (Bentley and Caudy, 1983; Caudy and Bentley, 1987). This could be because the band of epithelial cells within the trochanter promotes circumferential growth, or because the cells comprising the trochanter/coxa boundary and the region just proximal to it are non-permissive or aversive for growth cone migration, or both. The extension of many axon branches across the trochanter/coxa boundary following treatment with MAb 6F8 suggests that the trochanter/coxa boundary cells, which do not express fasciclin IV, are not aversive or non-permissive. Thus the change in behavior at the boundary appears to be due to the ability of fasciclin IV expressing epithelial cells to promote circumferential extension of processes from the Ti1 growth cones.

Secondly, treatment with MAb 6F8 results in frequent defasciculation of the axons of the two Ti1 neurons, and also formation of abnormal multiple axon branches, within the trochanter over fasciclin IV-expressing epithelial cells. Previous studies have shown that treatment with antibodies against ligands expressed on non-neural substrates (Landmesser et al., 1988), or putative competitive inhibitors of substrate ligands (Wang and Denburg, 1992) can promote defasciculation and increased axonal branching. Our results suggest that Ti1 axon:axon fasciculation and axon branching also are strongly influenced by interactions with substrate ligands, and that fasciclin IV appears to be a component of this interaction within the trochanter.

Thirdly, despite the effects of MAb 6F8 on axon branching, and on crossing the trochanter/coxa boundary, there remains a pronounced tendency for branches to grow ventrally both within the trochanter and within the distal region of the coxa. Consequently, all signals which can promote ventral migration of the growth cones have not been blocked by MAb 6F8 treatment. Antibody treatment may have a threshold effect in which ventral growth directing properties of fasciclin IV are more robust, and less incapacitated by treatment, than other features; alternatively, guidance information promoting ventral migration may be independent of fasciclin IV. Time lapse video experiments to determine how the abnormal pathways we observe actually form can resolve these issues.

These results demonstrate that fasciclin IV functions as a guidance cue for the Ti1 growth cones just distal to the trochanter/coxa boundary, is required for these growth cones to stop proximal growth and spread circumferentially, and that the function of fasciclin IV in Ti1 pathway formation result from interactions between a receptor/ligand on the Ti1 growth cones and fasciclin IV on the surface of the band of epithelial cells results in changes in growth cone morphology and subsequent reorientation. Fasciclin IV appears to elicit this change in growth cone morphology and orientation via regulation of adhesion, a signal transduction function, or a combination of the two.

EXPERIMENTAL PROCEDURES

Immunocytochemistry

Grasshopper embryos were obtained from a colony maintained at the U.C. Berkeley and staged by percentage of total embryonic development (Bentley et al., 1979). Embryos were dissected in PBS, fixed for 40 min in PEM-FA [0.1 M PIPES (pH6.95), 2.0 mM EGTA, 1.0 mM MgSO₄, 3.7% formaldehyde], washed for 1 hr with three changes in PBT (1×PBS, 0.5% Triton X-100, 0.2% BSA), blocked for 30 min in PBT with 5% normal goat serum, and incubated overnight at 4° C. in primary antibody. PBSap (1×PBS, 0.1% Saponin, 0.2% BSA) was used in place of PBT with MAb 8G7. Antibody dilutions were as follows: MAb 6F8 1:1, polyclonal antisera directed against a fasciclin IV bacterial fusion protein (#98-3) 1:400; MAb 8G7 1:4; MAb 8C6 1:1. The embryos were washed for one hour in PBT with three changes, blocked for 30 min, and incubated in secondary antibody for at least 2 hr at room temperature. The secondary antibodies were HRP-conjugated goat anti-mouse and anti-rat IgG (Jackson Immunoresearch Lab), and were diluted 1:300. Embryos were washed in PBT for one hour with three changes and then reacted in 0.5% diaminobenzidine (DAB) in PBT. The reaction was stopped with several washes in PBS and the embryos were cleared in a glycerol series (50%, 70%, 90%), mounted and viewed under Nomarski or bright field optics. For double-labelled preparations the first HRP reaction was done in PBT containing 0.06% NiCl, followed by washing, blocking, and incubation overnight in the second primary antibody. The second antibody was visualized with a DAB reaction as described above. Embryos cultured in the presence of monoclonal antibodies were fixed and incubated overnight in goat anti-HRP (Jackson Immunoresearch Labs) conjugated to RITC (Molecular Probes), washed for one hour in PBT with three changes, mounted in 90% glycerol, 2.5% DABCO (Polysciences), and viewed under epifluorescence. S2 cells were stained with polyclonal sera #98-3 diluted 1:400 and processed as described previously (Snow et al., 1989).

MONOCLONAL ANTIBODY BLOCKING EXPERIMENTS

In order to test for functional blocking, monoclonal antibody reagents were prepared as follows. Hybridoma supernatant was brought to 20% with H₂O-saturated (NH₄)₂SO₄, incubated in ice 1 hr, and spun at 15,000 g at 4° C. for 20 min. The supernatant was brought to 56% with H₂O-saturated (NH₄)₂SO₄, incubated overnight at 4° C., spun as above. The pellet was resuspended in PBS using approximately 1/40 volume of the original hybridoma supernatant (often remaining a slurry) and dialyzed against 1×PBS overnight at 4° C. with two changes. This reagent is referred to as “concentrated hybridoma supernatant.” Purified IgG was obtained by using Immunopure Plus Immobilized Protein A IgG Purification Kit (Pierce) to isolate IgG from the concentrated hybridoma supernatant. Fab fragments were obtained using the ImmunoPure Fab Preparation Kit (Pierce) from the previously isolated IgGs. For blocking experiments each reagent was diluted into freshly made supplemented RPMI culture media (O'Connor et al., 1990) and dialyzed overnight at 4° C. against 10 volumes of the same culture media. Dilutions were as follows: concentrated hybridoma supernatant 1:4; purified IgG 150 mg/ml; Fab 75 mg/ml.

Embryos for culture experiments were carefully staged to between 31 and 32% of development. As embryos in each clutch typically differ by less that 1% of embryonic development from each other, the growth cones of the Ti1 neurons at the beginning of the culture period were located approximately in the mid-femur, well distal to the trochanter/coxa segment boundary. From each clutch at least two limbs were filleted and the Ti1 neurons labelled with the lipophillic dye Di I (Molecular Probes) as described (O'Connor et al., 1990) in order to confirm the precise location of the Ti1 growth cones. Prior to culturing, embryos were sterilized and dissected (Chang et al., 1992). The entire amnion and dorsal membrane was removed from the embryo to insure access of the reagents during culturing. Embryos were randomly divided into groups and cultured in one of the blocking reagents described above. Cultures were incubated with occasional agitation at 30° C. for 30 hrs. At the end of the culture period embryos were fixed and processed for analysis as described above in immunocytochemistry.

For each culture experiment, the scoring of the Ti1 pathway in each limb was confirmed independently by a second observer. There was no statistically significant variation between the two observers. Limbs from MAb cultured embryos were compared to representative normal limbs from non-MAb cultured embryos and were scored as abnormal if any major deviation from the normal Ti1 pathway was observed. The Ti1 pathway was scored as abnormal for one or more of the following observed characteristics: (1) defasciculation for a minimum distance of approximately 25 mm anywhere along the pathway, (2) multiple axon branches that extended ventrally within the trochanter, (3) presence of one or more axon branches that crossed the trochanter/coxa boundary dorsal to the Cx1 cells, but then turned ventrally in the coxa and contacted the Cx1 cells, (4) the presence of axon branches that crossed the trochanter/coxa segment boundary, did not turn ventrally, but continued proximally toward the CNS, and (5) failure of ventrally extended axons within the trochanter to contact and reorient proximally to the Cx1 cells. For each MAb tested, the data are presented as a percentage of the abnormal Ti1 pathways observed.

Protein Affinity Purification and Microsequencing

Grasshopper fasciclin IV was purified by passing crude embryonic grasshopper lysate (Bastiani et al., 1987) over an Affi-Gel 15 column (Bio Rad) conjugated with the monoclonal antibody 6F8. Protein was eluted with 50 mM DEA (pH 11.5), 0.1% Lauryldimethylamine oxide (Cal Bio Chem), and 1 mM EDTA. Protein was then precipitated, denatured, modified at cysteines, and digested with either trypsin or Lys-C (Boehringer-Mannheim). Individual peptides were resolved by RP-HPLC and microsequenced (Applied Biosystems 4771 Microsequencer) using standard chemistry.

PCR Methods

DNA complementary to poly(A)+RNA from 45%-50% grasshopper embryos was prepared (Sambrook et al., 1989). PCR was performed using Perkin Elmer Taq polymerase (Saiki et al., 1988), and partially degenerate (based on grasshopper codon bias) oligonucleotides in both orientations corresponding to a portion of the protein sequence of several fasciclin IV peptides as determined by microsequencing. These oligonucleotides were designed so as not to include all of the peptide-derived DNA sequence, leaving a remaining 9-12 base pairs that could be used to confirm the correct identity of amplified products. All possible combinations of these sequences were tried. 40 cycles were performed, the parameters of each cycle as follows: 96° for one min; a sequentially decreasing annealing temperature (2° C./cycle, starting at 65° C. and ending at 55° C. for remaining 35 cycles) for 1 min; and at 72° C. for one min. Reaction products were cloned into the Sma site of M13 mp10 and sequenced. Two products, 1074 bp and 288 bp in length, contained DNA 3′ to the oligonucleotide sequences encoded the additional amino acid sequence of the fasciclin IV peptide from which the oligonuceotides were derived. These two fragments have one end in common, and the oligonucleotides used to amplify them correspond to the anino acid sequences Met-Tyr-Val-Gln-Phe-Gly-Glu-Glu and Met-Asp-Glu-Ala-Val-Pro-Ala-Phe (fasciclin IV residue 29-386) (SEQ ID NO:58), and His-Thr-Leu-Met-Asp-Glu-Ala and Lys-Asn-Tyr-Val-Val-Arg-Met-Asp-Glu (fasciclin IV residue 376-472) (SEQ ID NO:58).

cDNA Isolation and Sequence Analysis

Both PCR products were used to screen 1×10⁶ clones from a grasshopper embryonic cDNA library (Snow et al., 1988). 21 clones that hybridized to both fragments were recovered, and one 2600 bp clone was sequenced using the dideoxy. chain termination method (Sanger et al., 1977) and Sequenase (US Biochemical Corp.). Templates were made from M13 mp10 vectors containing inserts generated by sonication of plasmid clones. One cDNA was completely sequenced on both strands using Oligonucleotides and double strand sequencing of plasmid DNA (Sambrook et al., 1989) to fill gaps. Two additional cDNAs were analyzed by double strand sequencing to obtain the 3′ 402 bp of the transcript. All three cDNAs were used to construct a plasmid containing the entire transcript. The complete transcript sequence is 2860 bp in length with 452 bp of 5′ and 217 bp of 3′ untranslated sequences containing stop codons in all reading frames. The predicted protein sequence was analyzed using the FASTDB and BLASTP programs (Intelligenetics). The fasciclin IV ORF unambiguously contains 10 of the 11 peptide sequences determined by microsequencing the fasciclin IV trypsin and Lys-C peptides.

Generation of Polyclonal Antibodies From Bacterial Fusion Proteins

Bacterial trpE fusion proteins were constructed using pATH (Koerner et al., 1991) vectors, three restriction fragments encoding extracellular sequences, and one fragment (770 bp HindIII/Eco R1, which includes amino acids 476-730) encoding both extracellular and intracellular sequences (designated #98-3). Fusion proteins were isolated by making an extract of purified inclusion bodies (Spindler et al., 1984), and rats were immunized with ˜70 mg of protein emulsified in RIBI adjuvant (Immunochem Research). Rats were injected at two week intervals and serum was collected 7 days following each injection. Sera were tested histologically on grasshopper embryos at 45% of development. Construct #98-3 showed a strong response and exhibited a staining pattern identical to that of MAb 6F8. Two of the extracellular constructs responded weakly but also showed the fasciclin IV staining pattern. All pre-immune sera failed to stain grasshopper embryos.

S2 Cell Transfections, Aggregation Assays, and Western Analysis

A restriction fragment containing the full length fasciclin IV cDNA was cloned into pRmHa-3 (Bunch et al, 1988) and co-transformed into Drosophila S2 cells (Schneider, 1972) with the plasmid pPC4 (Jokerst et al., 1989), which confers a-amanitin resistance. S2 cells were transformed using the Lipofectin Reagent and recommended protocol (BRL) with minor modifications. All other S2 cell manipulations are essentially as described (Snow et al.,1989), including adhesion assays. Fasciclin IV expression in transformed cell lines was induced for adhesion assays and histology by adding CuSO₄ to 0.7 mM and incubating for at least 48 hrs. Northern analysis confirmed transcription of fasciclin IV and surface-associated staining of the S2 cells with polyclonal serum #98-3 strongly suggests fasciclin IV is being transported to the cell surface. Preparation of membranes from S2 cells and from grasshopper embryos, PAGE, and Western blot were performed as previously described (Elkins et al., 1990b) except that signal was detected using the enhanced chemiluminescence immunodetection system kit (Amersham). Amount of protein per lane in each sample loaded: fasciclin IV protein, ˜5 ng; S2 cell membranes, 40 mg; grasshopper membranes 80 mg. Amounts of protein loaded were verified by Ponceau S staining of the blot prior to incubation with the antibody.

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Genbank Accession Number: The accession number for the sequence reported in this paper is L00709.

II. Isolation and Characterization of Tribolium (SEQ ID NOS:63 & 64) and Drosophila (SEQ ID NOS:59 & 60) Semaphorin I, Drosophila Semaphorin II (SEQ ID NOS:61 & 62) Human Semaphorin III (SEQ ID NOS:53 & 54) and Vaccinia Virus Semaphorin IV (SEQ ID NOS:55 & 56) and Variola Major (smallpox) Virus Semaphorin V (SEQ ID NOS:67 & 68)

We used our G-Semaphorin I cDNA in standard low stringency screening methods (of both cDNA and genomic libraries) in an attempt to isolate a potential Semaphorin I homologue from Drosophila. We were unsuccessful in these screens. Since the sequence was novel and shared no similarity to anything else in the data base, we then attempted to see if we could identify a Semaphorin I homologue in other, more closely related insects. If possible, we would then compare these sequences to find the most conserved regions, and then to use probes (i.e., oligonucleotide primers for PCR) based on these conserved regions to find a Drosophila homologue.

In the process, we used the G-Semaphorin I cDNA in low stringency screens to clone Semaphorin I cDNAs from libraries made from locust Locusta migratoria embryonic RNA and from a cDNA embryonic library from the cricket Acheta domestica. We used PCR to clone genomic fragments from genomic DNA in the beetle Triboliwn, and from the moth Manduca. We then used the Triboliun genomic DNA fragment to isolate cDNA clones and ultimately sequenced the complete ORF for the Tribolium cDNA.

In the meantime, we used the partial Tribolium and Manduca sequences in combination with the complete grasshopper sequence to identify conserved regions that allowed us to design primers for PCR in an attempt to clone a Drosophila Semaphorin I homologue. Several pairs of primers generated several different bands, which were subcloned and sequenced and several of the bands gave partial sequences of the Drosophila Semaphorin homologue. One of the bands gave a partial sequence of what was clearly a different, more divergent gene, which we call D-Semaphorin II.

Based on the sequence of PCR products, we knew we had identified two different Drosophila genes, one of which appeared to be the Semaphorin I homologue, and the other a second related gene. The complete ORF sequence of the D-Semaphorin I homologue revealed an overall structure identical to G-Semaphorin I: a signal sequence, an extracellular domain of around 550 amino acids containing 16 cysteines, a transmembrane domain of 25 amino acids, and a cytoplasmic domain of 117 amino acids. When we had finished the sequence for D-Semaphorin II, we were able to begin to run homology searches in the data base, which revealed some of its structural features further described herein. The Semaphorin II sequence revealed a different structure: a signal sequence of 16 amino acids, a ˜525 amino acid domain containing 16 cysteines, with a single immunoglobulin (Ig) domain of 66 amino acids, followed by a short unique region of 73 amino acids. There is no evidence for either a transmembrane domain or a potential phospholipid linkage in the C-terminus of this protein. Thus, it appears that the D-Semaphorin II protein is secreted from the cells that produce it. The grasshopper, Tribolium, and Drosophila Semaphorin I cDNA sequences, as well as the sequence of the D-Semaphorin II cDNA, are shown herein. In addition, we used this same technique to identify Semaphorin I genes in a moth, Manduca sexta, a locust, Locusta migratoria, and a cricket, Acheta domestica.

With this large family of insect Semaphorin genes, we identified a number of good stretches of the right amino acids (with the least degeneracy based on their codons) with strong homology for designing primers for PCR to look for human genes. We designed a set of oligonucleotide primers, and plated out several human cDNA libraries: a fetal brain library (Stratagene), and an adult hippocampus library. We ultimately obtained a human cDNA PCR bands of the right size that did not autoprime and thus were good candidates to be bonafide Semaphorin-like cDNAs from humans. These bands were purified, subcloned, and sequenced.

Whole-mount in situ hybridization experiments showed that D-Semaphorin I and II are expressed by different subsets of neurons in the embryonic CNS. D-Semaphorin I is expressed by certain cells along the midline as well as by other neurons, whereas D-Semaphorin II is not expressed at the midline, but is expressed by a different subset of neurons. In addition, D-Semaphorin II is expressed by a subset of muscles prior to and during the period of innervation by specific motoneuron. On the polytene chromosomes, the D-Semaphorin I gene maps to (gene-band-chromosome) 29E1-22L and that of D-Semaphorin II to 53C9-102R. We have identified loss of function mutations in the D-Semaphorin I gene and a pair of P-element transposon insertions in the D-Semaphorin II gene which appear to cause severe phenotypes.

When we lined up the G-Semaphorin I, T-Semaphorin I, D-Semaphorin I, and D-Semaphorin II sequences and ran the sequences through a sequence data base in search of other sequences with significant similarity, we discovered a curious finding: these Semaphorins share sequence similarity with the A39R open reading frame (ORF) from Vaccinia virus and the A43R ORF from Variola Major (smallpox) virus and we discovered that the amino acids shared with the virus ORF were in the same regions where the insect proteins shared their greatest similarity. The viral ORF began with a putative signal sequence, continued for several hundred amino acids with sequence similarity to the Semaphorin genes, and then ended without any membrane linkage signal (suggesting that the protein as made by the infected cell would likely be secreted).

We reasoned that the virus semaphorins were appropriated host proteins advantageously exploited by the viruses, which would have host counterparts that most likely function in the immune system to inhibit or decrease an immune response, just as in the nervous system they appear to function by inhibiting growth cone extension. Analogous to situations where viruses are thought to encode a secreted form of a host cellular receptor, here the virus may cause the infected cell to make a lot of the secreted ligand to mimic an inhibitory signal and thus help decrease the immune response.

III. Isolation and Characterization of Murine CNS Semaphorin III Receptor Using Epitope Tagged Human Semaphorin III (hSIII)

mRNA was isolated from murine fetal brain tissue and used to construct a cDNA library in a mammalian exprssion vector, pCMX, essentially as in Davis et al. (1991) Science 253, 59.

The transfection and screening procedure is modified from Lin et al (1992) Cell 68, 775. COS cells grown on glass slide flaskettes are transfected with pools of the cDNA clones, allowed to bind radioiodinated hSIII truncated at the C-terminus end of the semaphorin domain. In parallel, similarly treated COS cells are allowed to bind unlabelled human semaphorin III truncated at the C-terminus end of the semaphorin domain and there joined to a 10-amino acid extension derived from the human c-myc proto-oncogene product. This modified hSIII allows the identification of hSIII receptors with the use of the tagged ligand as a bridge between the receptor and a murine monoclonal antibody which is specific for an epitope in the c-myc tag. Accordingly, after binding unlabelled hSIII the cells are exposed to the monoclonal which may be labeled directly or subsequently decorated with a secondary anti-mouse labeled antibody for enhanced signal amplification.

Cells are then fixed and screened using dark-field microscopy essentially as in Lin et al. (supra). Positive clones are identified and sequence analysis of murine CNS Semphorin III receptor cDNA clones by the dideoxy chain termination method is used to construct full-length receptor coding sequences.

It is evident from the above results that one can use the methods and compositions disclosed here in for making and identifying diagnostic probes and therapeutic drugs. It will also be clear to one skilled in the art from a reading of this disclosure that advantage can be taken to effect alterations of semaphorin responsiveness in a host.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

TABLE 1 Deduced amino acid sequence of semaphorin gene family. Approximate position of enumerated peptide classes are indicated by parenthetical (a) through (o); semaphorin domains are bounded by arrows; G: grasshopper semaphorin I (SEQ ID NO:58), T: Tribolium semaphorin I (SEQ ID NO:64), D1: Drosophila semaphorin I (SEQ ID NO:60), D2: Drosophila semaphorin II (SEQ ID NO:62), H3: Human semaphorin I (SEQ ID ON:54), V4: Vaccinia virus semaphorin IV (SEQ ID NO:56), V5: Variola virus (human small pox) semaphorin IV (SEQ ID NO:66); small case residues: conserved residues; underline: signal sequence; solid bar: transmembrane domain; double dashes: immunoglobulin domain. G                    MRAALVAVAALLWVVALHAAAWVNDVSPKMYVQFGEERVQR T                       MVVKILVWSICLIALCHAWMPDSSSKLINHFKSVESKS D1 D2 MSLLQLSPLLALLLLLCSSVSETAADYENTWNFYYERPCCTGNDQGNNNYGKHGADHVRE H3              MGWLTRIVCLFWGVLLTARANYQNGKNNVPRLKLSYKEMLESNNVIT V4                                 MMVLLHAVYSIVFVDVIIIKVQRYINDI G f-Lg--nESHKDHfKLLeKDHNSlLvga--rNI---VYnISLRDLTEFTEQRiEwHSsGAHRELcY T fT-g--nATFPDHfIVLNQDETSILvgG--rNR---VYnLSIFDLSERKGGRiDwPSsDAHGQLcI D1 D2 fNCgKLY---YRTfHMNeDRDT-lYvgaMDrVF---RVnLQNISSSNCNRDAiNLEPTRDDVVScV H3 fN-gLAnSSSYHTfLLDeERSR-lYvgaKDHIFSFDLVnI-------KDFQKiVwPVsYTRRDEcK V4 LTLDIFYLFKKMIPLLFILFYFANGIEWHKFETSEEIISTYLLDDV------LYTGVNGAVYTFSN                              A              F               V        DDCQN-YIR(a)          ICGTN(b) G LkgkS-Eddqqn-yir--VlAKIDDDrVLIcgtnaYKpLcRHyALKd-----GDyVVeKEYEgRg---- T LkgkT-Dddcqn-yir--ilYSSEPGKlVIcgtnSYKpLcRTyAFKE-----GKyLVeKEVEgIg---- D1       Eddcqn-yir--iMVVPSPGrlFvcgtnSFRpMcNTyIISd-----SNyTLeATKNgQA---- D2 SkgkSQIFdcKnHViQ--SMDQGD--rlYvcgtnaHNpKDYViYANL-----THLPRSEYVIgVgLGIA H3 WAgkDILKEcAn-FiK--VlKAYNQTHlYAcgtGaFHpIcTYiEIGHPEDNIFKLENSHFENgRg---- V4 NkLNKTGLTNNn-yiTTSiKVEDADKDTLvcgtnNGNpKcWKiDGSd----------------------  S F                                   A  CPYDP(c)                             TVADFSG(d) G LcpFdpDh---------nstAIYSEgQ-------lysAtvadfsgTdpLiyrGpl------------ T LcpyNpEh---------nstsVSYNgQ-------lFsAtvadfsgGdpLiyrEpQ------------ D1 VcpydpRh---------nstsVLADNE-------lysgtvadfsgSdpIiyrEpl------------ D2 KcpydpLD---------nstAIYVENGNPGGLPGlysgtNaEfTKAdTViFrTDlYNTSAKRLEYKF H3 KSpydpKL---------LTAsLLIDgE-------lysgtAadfMgRdFAiFrT-lGHHHPIRTEQHD V4 ----dpKhRGRGYAPYQnsKVTIISHNGcYLSDINIsKEGIKRWRRFDGPcGYDl------------ V5                                                    MIYl------------                                   N            LNAPNFV(e)     (f)FFFRETA EYINCGK(g)      (h)DKGG G -rteRSdLkQ-lnapnfv-NTMEyNdFIFfffretaveyincgkaiysrvarvckHdkgg T -rteLSdLkQ-lnapnfv-NsVAygdYIFffYretaveyMncgkViysrvarvckDdkgg D1 -QteQYdSLS-lnapnfv-SsFtQgdFvyfffretaveFincgkaiysrvarvckWdkgg D2 KrtLKYdSkW-lDKpnfv-GsFDIgEYvyfffretaveyincgkaVysriarvckKdVgg H3 -SRWLNdpkF-ISaHLISESdNPEDdkvyfffreNaIDGEHSgkaTHAriGQIckndFgg V4 YTADNVIpkDGlRGA-fvDKdGty-dkvyILfTDtIG-SKRIVkIPy--iaQMcLndEgg V5 YTADNVIpkDGlQGA-fvDKdGty-dkvyILfTDtIG-SKRIVkIPy--iaQMcLndECg          SSY(i)     V PH      WTTFLKAR NCSIPG(j) G phQF-GDrwtsflkSrlncsVpgDypfyf---neiqs---tsdIIegNyGGQVEkliygv T phQ-SRDrwtsflkarlncsipgEypfyf---Deiqs---tsdIvegRyNsDDskIiygI D1 phRF-RNrwtsflkSrlncsipgDypfyf---neiqs---AsNLvegQyGsMSskliygv D2 KNLl-AhNwAtYlkarlncsiSgEFpfyf---neiqs---VYQL-----PsDKsRF-FAT H3 -hRSLVNKwttflkarlIcsVpgPNGIDTHf-DeLq------dVFLMNFKDPKNPVVygv V4 pSSlSShrwStflkVElEcDiDgRSYRQIIHSRTiKTDNDtILYvF--FDsPYsk----- V5 pSSlSShrwStLlkVElEcDiDgRSYRQINHSKTiKQIMIRYYMYSLIVLFQVRIMYLFY                                        V          GSAVC(k)                  NSNWLPV(l)    PRPGTCVND(m) G fttpVnSiGgsavcafsmKSiLESfDgPfkeqETMnsnwlAvPSLKvpeprpgQcvndsr T LttpVnAiGgsaIcayQmAdiLRVfEgSfkHqETInsnwlpvPQNLvpeprpgQcvRdsr D1 fNtpSnSiPgsavcafALQdiADTfEgQfkeqTGInsnwlpvNNAKvpDprpgScHndsr D2 fttSTnGLIgsavcSfHINEiQAAfNgKfkeqSSSnsAwlpvLNSRnpeprpgTcvndTS H3 fttSSnIFKgsavcMysmSdVRRVfLgPYAHRDGPnYQwVp-YQGRvpYprpgTc--PsK V4 ----------saLcTysmNTiKQSfSTSKLeg----------YTKQLpSpApgIcLPAGK V5 EYH G ---------TlpdVSVnfV-kShTlmdEAvpaFfTRpilIrIslQyrftKiAvdQqvRtPDgKAYdvLf T ---------IlpdKNVnfi-kThSlmED-vpaLfGKpVlVrVslQyrftAiTvdPqvKtINNQYLdvLY D1 ---------AlpdPTLnfi-kThSlmdENvpaFfSQpilVrTsTIyrftQiAvdAqIKtPGgKTYdvIf D2 ---------NlpdTVLnfi-RShPlmdKAvNHEHnNpVYYKRDlVFTK-LVVDKIRIDILNQEYI-vYY H3 TFGGFDSTKDlpdDVITfA-rshPAmYNPvFPMNnRpiVIKTDVNyQftQiVvd-RvDAEDgQY-dvMf V4 ---------VVpHTTFEViEKYNVlDdIIKp-LSnQpiFEGPSGVKWFDIKEKENEHREYRIYFIKENS G igtddgkvIkALnSAsFDSSDTvDSvVIeeLQvLPPGVpVKnlYVvr-------Mdg--d T igtddgkvLkAvnIPKRHAKALLYRKYRTSVHPHGA--pVKQlKIAP------------G D1 VgtdHgkIIkSvnAEsADSADKvTSvVIeeIDvLTKSEpIRnlEIvrTMQYDQPKdgSYd D2 VgtNLgRIYkIvnGEsLSKLLDIFEvAPNeAIQVMEISQTR------------------- H3 igtdVgTvLkVvSIPKETWY-DLEEvLLeeMTvFREPTAISA------------MELSTK V4 iYSFdTkSKQTRSSQVDARLFSvMVTSKPLFIADIGIGVGMPQMKKILKM*                                    DPYCAWD(n) G dSklVVvSdDEiLAiKlhrcGSdkItNcRecvSlqdpycawdNVELKcTAVgSpDwSAG T YGkVVVvGKDEiRLANlNHcAS-k-tRcKDcvElqdpHcawdAKQNLcVSIDTVTSY-- D1 dGklIivTdSQVVAiQlhrcHNdkItScSecvAlqdpycawdKIAGKcRSHgApRw-LE D2 -KSlYiGTdHRiKQiDlAMc-NRRYDNcFRcv--RdpycGwdKEANTcRPY-------- H3 QQQlYiGSTAGVAQLPlhrcDIYG-KAcAecCLARdpycawd--GSAcS---RYFPTAK                         ↓ G KrRFIqNISLgEH-KAcGGRPQTEIVASPVPTQPTTKSSGDPVHSIHQAEFEpeiDNEiVI T -rFLIqdvVRgDD-NKcWsPQTDKkTVIKNKPSEVENEITNSIDEKDLDsSdpLiKTGLdD D ENYFYqNvATgQH-AAcPsGKINSkDANAGEGKGFRNDMDLLDSRRQ--sKdQeiIDNidK D2 ELDLLqdvANETS-DIcDsSVLKKk H3 RrTRRqdIRNgDPLTHcSDLHDNHH G GVddSNVIPNTLAEINHAGSKLPSSQEKlPiytaetlTiaIvTSCLGAlVvgfIsgFLFS T DSdcDPVSENSIGGcAV--------RQQlViytaGtlHiVvvVVsiVglFSWLYsgLSVF D NFEdD---------------------IINAQytVetlVMavLAGsiFSlLvgfFTgYFCG G rrcRGEDYTDMpFpdQRHQLNRLTEAGlNADsPYLPPCANnkAAInlvLNv-----PpkN T AKFHSd----SQypEAPFIEQHNHLERlsANQTGYLTPRAnk-AVnlvvKvSSSTPRpkK D rrcHKdEDDNLpypdTEYEYFEQRQNVNsFPsSCRIQQEPKLLPQVEEvTYAEPVLLpQP                     KKTYI(o) G AnGKNANsSAENKP----IQkktyi* T DnLDVSKDLNIASDGTLQKIkktyi* D PPPNKMHsPKNTLRKPPMHQMHQGPNSETLFQFHVTATTPSSRIVVATTSEHCVPTR* D2                 -----IVVTyg---QsVHlGcFVkIPEVlKNEQvTwYHHSKDKG H3                 GHSPEERIIygVENSsTFlEcSPkSQRAl----vYwQFQRRNEE                                 ============================ D2 rYeIRYSPTKYiETtERglVVVsVNEAdGgRyDchLGGSLLcSYNITVDAHRcTPPNKSN H3 rKeE-IRVDDHiIRtDQglLLRsLQQKdSgNyLchAVEHGFIQTLLKVTLEVIDTEHLEE ========================================= D2 DYQKIYSDWcHEFEKYKTAMKSWEKKQGQcSTRQNFScNQHPNEIFRKPNV* H3 LLHKDDDGDGSKTKEMSNSMTPSQKVWYRDFMQLINHPNLNTMDEFcEQVWKRDRKQRRQ H3 RPGHTPGNSNKHLQENKKGRNRRTHEFERAPRSV*

100 6 amino acids amino acid single linear peptide Peptide 1..6 /label= SEQ01 /note= “Xaa denotes D or E at residue #1; Q,K,R,A or N at residue #3; and Y,F or V at residue #5” 1 Xaa Cys Xaa Asn Xaa Ile 1 5 6 amino acids amino acid single linear peptide Peptide 1..6 /label= SEQ02 /note= “Xaa denotes Q,K,R,A or N at residue #2; Y,F or V at residue #4; and R,K,Q or T at residue #6” 2 Cys Xaa Asn Xaa Ile Xaa 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ03 /note= “Xaa denotes N or G at residue #4; A,S or N at residue #5; Y,F,H or G at residue #6; and K,R,H,N or Q at residue #7” 3 Cys Gly Thr Xaa Xaa Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ04 /note= “Xaa denotes N or G at residue #4; and A,S or N at residue #5” 4 Cys Gly Thr Xaa Xaa Xaa Xaa Pro 1 5 10 amino acids amino acid single linear peptide Peptide 1..10 /label= SEQ05 /note= “Xaa denotes N or G at residue #4; and C or D at residue #10” 5 Cys Gly Thr Xaa Xaa Xaa Xaa Pro Xaa Xaa 1 5 10 13 amino acids amino acid single linear peptide Peptide 1..13 /label= SEQ06 /note= “Xaa denotes C or D at residue #10; and Y or I at residue #13” 6 Cys Gly Thr Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa 1 5 10 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ07 /note= “Xaa denotes R,I,Q or V at residue #1; G or A at residue #2; L,V or K at residue #3; C or S at residue #4; F or Y at residue #6; and D or N at residue #7” 7 Xaa Xaa Xaa Xaa Pro Xaa Xaa 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ08 /note= “Xaa denotes C or S at residue #1; F or Y at residue #3; D or N at residue #4; D,E,R or K at residue #6; and H,L or D at residue #7” 8 Xaa Pro Xaa Xaa Pro Xaa Xaa 1 5 9 amino acids amino acid single linear peptide Peptide 1..9 /label= SEQ09 /note= “Xaa denotes G or A at residue #3; C or S at residue #5; and D or N at residue #8” 9 Gly Xaa Xaa Xaa Xaa Pro Tyr Xaa Pro 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ10 /note= “Xaa denotes F or Y at residue #2; G or A at residue #4; and V,N or A at residue #6” 10 Leu Xaa Ser Xaa Thr Xaa Ala 1 5 9 amino acids amino acid single linear peptide Peptide 1..9 /label= SEQ11 /note= “Xaa denotes F or Y at residue #2; D or E at residue #8; and F or Y at residue #9” 11 Leu Xaa Ser Xaa Thr Xaa Ala Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ12 /note= “Xaa denotes F or Y at residue #1; G or A at residue #3; V,N or A at residue #5; D or E at residue #7; and F or Y at residue #8” 12 Xaa Ser Xaa Thr Xaa Ala Xaa Xaa 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ13 /note= “Xaa denotes N or D at residue #2; and A or K at residue #3” 13 Leu Xaa Xaa Pro Asn Phe Val 1 5 5 amino acids amino acid single linear peptide 14 Phe Phe Phe Arg Glu 1 5 6 amino acids amino acid single linear peptide Peptide 1..6 /label= SEQ15 /note= “Xaa denotes F or Y at residue #3; and T or N at residue #6” 15 Phe Phe Xaa Arg Glu Xaa 1 5 6 amino acids amino acid single linear peptide Peptide 1..6 /label= SEQ16 /note= “Xaa denotes T or N at residue #5” 16 Phe Phe Arg Glu Xaa Ala 1 5 6 amino acids amino acid single linear peptide Peptide 1..6 /label= SEQ17 /note= “Xaa denotes F or Y at residue #2; and T or N at residue #5” 17 Phe Xaa Arg Glu Xaa Ala 1 5 6 amino acids amino acid single linear peptide Peptide 1..6 /label= SEQ18 /note= “Xaa denotes F or Y at residue #4” 18 Tyr Phe Phe Xaa Arg Glu 1 5 6 amino acids amino acid single linear peptide Peptide 1..6 /label= SEQ19 /note= “Xaa denotes F or Y at residue #1; and F or Y at residue #4” 19 Xaa Phe Phe Xaa Arg Glu 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ20 /note= “Xaa denotes F or Y at residue #1; F or Y at residue #2; F or Y at residue #3; and T or N at residue #6” 20 Xaa Xaa Xaa Arg Glu Xaa Ala 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ21 /note= “Xaa denotes I or V at residue #1; F or Y at residue #2; F or Y at residue #4; and F or Y at residue #5” 21 Xaa Xaa Phe Xaa Xaa Arg Glu 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ22 /note= “Xaa denotes K,F or Y at residue #2; F or Y at residue #4; F,Y,I or L at residue #5; F,Y,I or L at residue #6; and F or Y at residue #7” 22 Asp Xaa Val Xaa Xaa Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ23 /note= “Xaa denotes V or I at residue #1; F or Y at residue #2; F,Y,I or L at residue #3; F,Y,I or L at residue #4; R or T at residue #6; and T or N at residue #8” 23 Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ24 /note= “Xaa denotes V or I at residue #1; F or Y at residue #2; F,Y,I or L at residue #3; F,Y,I or L at residue #4; F or Y at residue #5; R or T at residue #6; E,D or V at residue #7; and T or N at residue #8” 24 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ25 /note= “Xaa denotes F or Y at residue #2; and C or S at residue #5” 25 Glu Xaa Ile Asn Xaa Gly Lys 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ26 /note= “Xaa denotes F or Y at residue #1; and A,V or I at residue #7” 26 Xaa Ile Asn Cys Gly Lys Xaa 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ27 /note= “Xaa denotes V or I at residue #2; A or G at residue #3; R or Q at residue #4; and V or I at residue #5” 27 Arg Xaa Xaa Xaa Xaa Cys Lys 1 5 9 amino acids amino acid single linear peptide Peptide 1..9 /label= SEQ28 /note= “Xaa denotes V or I at residue #2; R or Q at residue #4; and V or I at residue #5” 28 Arg Xaa Xaa Xaa Xaa Cys Xaa Xaa Asp 1 5 13 amino acids amino acid single linear peptide Peptide 1..13 /label= SEQ29 /note= “Xaa denotes V,A or I at residue #3; and V,A or I at residue #8” 29 Gly Lys Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Cys Lys 1 5 10 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ30 /note= “Xaa denotes R,K or N at residue #1; T,A or S at residue #3; T,A or S at residue #4; F,Y or L at residue #5; and K or R at residue #7” 30 Xaa Trp Xaa Xaa Xaa Leu Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ31 /note= “Xaa denotes F or Y at residue #1; K or R at residue #3; A or S at residue #4; and N or I at residue #7” 31 Xaa Leu Xaa Xaa Arg Leu Xaa Cys 1 5 6 amino acids amino acid single linear peptide Peptide 1..6 /label= SEQ32 /note= “Xaa denotes N or I at residue #1; I or V at residue #4; and P or S at residue #5” 32 Xaa Cys Ser Xaa Xaa Gly 1 5 9 amino acids amino acid single linear peptide Peptide 1..9 /label= SEQ33 /note= “Xaa denotes T,A or S at residue #2; T,A or S at residue #3; F,Y or L at residue #4; and A,S,V,I or L at residue #7” 33 Trp Xaa Xaa Xaa Leu Lys Xaa Xaa Leu 1 5 11 amino acids amino acid single linear peptide Peptide 1..11 /label= SEQ34 /note= “Xaa denotes T,A or S at residue #2; and T,A or S at residue #3” 34 Trp Xaa Xaa Xaa Leu Lys Xaa Xaa Leu Xaa Cys 1 5 10 11 amino acids amino acid single linear peptide Peptide 1..11 /label= SEQ35 /note= “Xaa denotes T or S at residue #3” 35 Trp Xaa Xaa Xaa Leu Lys Xaa Xaa Leu Xaa Cys 1 5 10 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ36 /note= “Xaa denotes F or Y at residue #1; F or Y at residue #2; and N or D at residue #3” 36 Xaa Xaa Xaa Glu Ile Gln Ser 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ37 /note= “Xaa denotes F or Y at residue #1; F or Y at residue #3; F or Y at residue #4; F or Y at residue #5; and N or D at residue #6” 37 Xaa Pro Xaa Xaa Xaa Xaa Glu 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ38 /note= “Xaa denotes V,I or L at residue #4; and F or Y at residue #7” 38 Gly Ser Ala Xaa Cys Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ39 /note= “Xaa denotes V,I or L at residue #3; and F or Y at residue #6” 39 Ser Ala Xaa Cys Xaa Xaa Xaa Met 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ40 /note= “Xaa denotes N or A at residue #3; and P or A at residue #6” 40 Asn Ser Xaa Trp Leu Xaa Val 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ41 /note= “Xaa denotes V,L or I at residue #1; and E,D,Y,S or F at residue #3” 41 Xaa Pro Xaa Pro Arg Pro Gly 1 5 9 amino acids amino acid single linear peptide Peptide 1..9 /label= SEQ42 /note= “Xaa denotes V,L or I at residue #1; and R or A at residue #5” 42 Xaa Pro Xaa Pro Xaa Pro Gly Xaa Cys 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ43 /note= “Xaa denotes E,D,Y,S or F at residue #2; and T,Q or S at residue #7” 43 Pro Xaa Pro Arg Pro Gly Xaa Cys 1 5 6 amino acids amino acid single linear peptide Peptide 1..6 /label= SEQ44 /note= “Xaa denotes H,F or Y at residue #3; and A or G at residue #5” 44 Asp Pro Xaa Cys Xaa Trp 1 5 6 amino acids amino acid single linear peptide Peptide 1..6 /label= SEQ45 /note= “Xaa denotes H,F or Y at residue #2; and A or G at residue #4” 45 Pro Xaa Cys Xaa Trp Asp 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ46 /note= “Xaa denotes A or G at residue #5” 46 Asp Pro Xaa Cys Xaa Trp Asp 1 5 12 amino acids amino acid single linear peptide 47 Cys Xaa Xaa Xaa Xaa Asp Pro Xaa Cys Xaa Trp Asp 1 5 10 11 amino acids amino acid single linear peptide 48 Cys Xaa Xaa Xaa Asp Pro Xaa Cys Xaa Trp Asp 1 5 10 10 amino acids amino acid single linear peptide 49 Cys Xaa Xaa Asp Pro Xaa Cys Xaa Trp Asp 1 5 10 15 amino acids amino acid single linear peptide 50 Cys Xaa Xaa Cys Xaa Xaa Xaa Xaa Asp Xaa Xaa Cys Xaa Trp Asp 1 5 10 15 14 amino acids amino acid single linear peptide 51 Cys Xaa Xaa Cys Xaa Xaa Xaa Asp Xaa Xaa Cys Xaa Trp Asp 1 5 10 13 amino acids amino acid single linear peptide 52 Cys Xaa Xaa Cys Xaa Xaa Asp Xaa Xaa Cys Xaa Trp Asp 1 5 10 2601 base pairs nucleic acid double linear cDNA CDS 16..2331 53 GGAATTCCCT GCAGC ATG GGC TGG TTA ACT AGG ATT GTC TGT CTT TTC TGG 51 Met Gly Trp Leu Thr Arg Ile Val Cys Leu Phe Trp 1 5 10 GGA GTA TTA CTT ACA GCA AGA GCA AAC TAT CAG AAT GGG AAG AAC AAT 99 Gly Val Leu Leu Thr Ala Arg Ala Asn Tyr Gln Asn Gly Lys Asn Asn 15 20 25 GTG CCA AGG CTG AAA TTA TCC TAC AAA GAA ATG TTG GAA TCC AAC AAT 147 Val Pro Arg Leu Lys Leu Ser Tyr Lys Glu Met Leu Glu Ser Asn Asn 30 35 40 GTG ATC ACT TTC AAT GGC TTG GCC AAC AGC TCC AGT TAT CAT ACC TTC 195 Val Ile Thr Phe Asn Gly Leu Ala Asn Ser Ser Ser Tyr His Thr Phe 45 50 55 60 CTT TTG GAT GAG GAA CGG AGT AGG CTG TAT GTT GGA GCA AAG GAT CAC 243 Leu Leu Asp Glu Glu Arg Ser Arg Leu Tyr Val Gly Ala Lys Asp His 65 70 75 ATA TTT TCA TTC GAC CTG GTT AAT ATC AAG GAT TTT CAA AAG ATT GTG 291 Ile Phe Ser Phe Asp Leu Val Asn Ile Lys Asp Phe Gln Lys Ile Val 80 85 90 TGG CCA GTA TCT TAC ACC AGA AGA GAT GAA TGC AAG TGG GCT GGA AAA 339 Trp Pro Val Ser Tyr Thr Arg Arg Asp Glu Cys Lys Trp Ala Gly Lys 95 100 105 GAC ATC CTG AAA GAA TGT GCT AAT TTC ATC AAG GTA CTT AAG GCA TAT 387 Asp Ile Leu Lys Glu Cys Ala Asn Phe Ile Lys Val Leu Lys Ala Tyr 110 115 120 AAT CAG ACT CAC TTG TAC GCC TGT GGA ACG GGG GCT TTT CAT CCA ATT 435 Asn Gln Thr His Leu Tyr Ala Cys Gly Thr Gly Ala Phe His Pro Ile 125 130 135 140 TGC ACC TAC ATT GAA ATT GGA CAT CAT CCT GAG GAC AAT ATT TTT AAG 483 Cys Thr Tyr Ile Glu Ile Gly His His Pro Glu Asp Asn Ile Phe Lys 145 150 155 CTG GAG AAC TCA CAT TTT GAA AAC GGC CGT GGG AAG AGT CCA TAT GAC 531 Leu Glu Asn Ser His Phe Glu Asn Gly Arg Gly Lys Ser Pro Tyr Asp 160 165 170 CCT AAG CTG CTG ACA GCA TCC CTT TTA ATA GAT GGA GAA TTA TAC TCT 579 Pro Lys Leu Leu Thr Ala Ser Leu Leu Ile Asp Gly Glu Leu Tyr Ser 175 180 185 GGA ACT GCA GCT GAT TTT ATG GGG CGA GAC TTT GCT ATC TTC CGA ACT 627 Gly Thr Ala Ala Asp Phe Met Gly Arg Asp Phe Ala Ile Phe Arg Thr 190 195 200 CTT GGG CAC CAC CAC CCA ATC AGG ACA GAG CAG CAT GAT TCC AGG TGG 675 Leu Gly His His His Pro Ile Arg Thr Glu Gln His Asp Ser Arg Trp 205 210 215 220 CTC AAT GAT CCA AAG TTC ATT AGT GCC CAC CTC ATC TCA GAG AGT GAC 723 Leu Asn Asp Pro Lys Phe Ile Ser Ala His Leu Ile Ser Glu Ser Asp 225 230 235 AAT CCT GAA GAT GAC AAA GTA TAC TTT TTC TTC CGT GAA AAT GCA ATA 771 Asn Pro Glu Asp Asp Lys Val Tyr Phe Phe Phe Arg Glu Asn Ala Ile 240 245 250 GAT GGA GAA CAC TCT GGA AAA GCT ACT CAC GCT AGA ATA GGT CAG ATA 819 Asp Gly Glu His Ser Gly Lys Ala Thr His Ala Arg Ile Gly Gln Ile 255 260 265 TGC AAG AAT GAC TTT GGA GGG CAC AGA AGT CTG GTG AAT AAA TGG ACA 867 Cys Lys Asn Asp Phe Gly Gly His Arg Ser Leu Val Asn Lys Trp Thr 270 275 280 ACA TTC CTC AAA GCT CGT CTG ATT TGC TCA GTG CCA GGT CCA AAT GGC 915 Thr Phe Leu Lys Ala Arg Leu Ile Cys Ser Val Pro Gly Pro Asn Gly 285 290 295 300 ATT GAC ACT CAT TTT GAT GAA CTG CAG GAT GTA TTC CTA ATG AAC TTT 963 Ile Asp Thr His Phe Asp Glu Leu Gln Asp Val Phe Leu Met Asn Phe 305 310 315 AAA GAT CCT AAA AAT CCA GTT GTA TAT GGA GTG TTT ACG ACT TCC AGT 1011 Lys Asp Pro Lys Asn Pro Val Val Tyr Gly Val Phe Thr Thr Ser Ser 320 325 330 AAC ATT TTC AAG GGA TCA GCC GTG TGT ATG TAT AGC ATG AGT GAT GTG 1059 Asn Ile Phe Lys Gly Ser Ala Val Cys Met Tyr Ser Met Ser Asp Val 335 340 345 AGA AGG GTG TTC CTT GGT CCA TAT GCC CAC AGG GAT GGA CCC AAC TAT 1107 Arg Arg Val Phe Leu Gly Pro Tyr Ala His Arg Asp Gly Pro Asn Tyr 350 355 360 CAA TGG GTG CCT TAT CAA GGA AGA GTC CCC TAT CCA CGG CCA GGA ACT 1155 Gln Trp Val Pro Tyr Gln Gly Arg Val Pro Tyr Pro Arg Pro Gly Thr 365 370 375 380 TGT CCC AGC AAA ACA TTT GGT GGT TTT GAC TCT ACA AAG GAC CTT CCT 1203 Cys Pro Ser Lys Thr Phe Gly Gly Phe Asp Ser Thr Lys Asp Leu Pro 385 390 395 GAT GAT GTT ATA ACC TTT GCA AGA AGT CAT CCA GCC ATG TAC AAT CCA 1251 Asp Asp Val Ile Thr Phe Ala Arg Ser His Pro Ala Met Tyr Asn Pro 400 405 410 GTG TTT CCT ATG AAC AAT CGC CCA ATA GTG ATC AAA ACG GAT GTA AAT 1299 Val Phe Pro Met Asn Asn Arg Pro Ile Val Ile Lys Thr Asp Val Asn 415 420 425 TAT CAA TTT ACA CAA ATT GTC GTA GAC CGA GTG GAT GCA GAA GAT GGA 1347 Tyr Gln Phe Thr Gln Ile Val Val Asp Arg Val Asp Ala Glu Asp Gly 430 435 440 CAG TAT GAT GTT ATG TTT ATC GGA ACA GAT GTT GGG ACC GTT CTT AAA 1395 Gln Tyr Asp Val Met Phe Ile Gly Thr Asp Val Gly Thr Val Leu Lys 445 450 455 460 GTA GTT TCA ATT CCT AAG GAG ACT TGG TAT GAT TTA GAA GAG GTT CTG 1443 Val Val Ser Ile Pro Lys Glu Thr Trp Tyr Asp Leu Glu Glu Val Leu 465 470 475 CTG GAA GAA ATG ACA GTT TTT CGG GAA CCG ACT GCT ATT TCA GCA ATG 1491 Leu Glu Glu Met Thr Val Phe Arg Glu Pro Thr Ala Ile Ser Ala Met 480 485 490 GAG CTT TCC ACT AAG CAG CAA CAA CTA TAT ATT GGT TCA ACG GCT GGG 1539 Glu Leu Ser Thr Lys Gln Gln Gln Leu Tyr Ile Gly Ser Thr Ala Gly 495 500 505 GTT GCC CAG CTC CCT TTA CAC CGG TGT GAT ATT TAC GGG AAA GCG TGT 1587 Val Ala Gln Leu Pro Leu His Arg Cys Asp Ile Tyr Gly Lys Ala Cys 510 515 520 GCT GAG TGT TGC CTC GCC CGA GAC CCT TAC TGT GCT TGG GAT GGT TCT 1635 Ala Glu Cys Cys Leu Ala Arg Asp Pro Tyr Cys Ala Trp Asp Gly Ser 525 530 535 540 GCA TGT TCT CGC TAT TTT CCC ACT GCA AAG AGA CGC ACA AGA CGA CAA 1683 Ala Cys Ser Arg Tyr Phe Pro Thr Ala Lys Arg Arg Thr Arg Arg Gln 545 550 555 GAT ATA AGA AAT GGA GAC CCA CTG ACT CAC TGT TCA GAC TTA CAC CAT 1731 Asp Ile Arg Asn Gly Asp Pro Leu Thr His Cys Ser Asp Leu His His 560 565 570 GAT AAT CAC CAT GGC CAC AGC CCT GAA GAG AGA ATC ATC TAT GGT GTA 1779 Asp Asn His His Gly His Ser Pro Glu Glu Arg Ile Ile Tyr Gly Val 575 580 585 GAG AAT AGT AGC ACA TTT TTG GAA TGC AGT CCG AAG TCG CAG AGA GCG 1827 Glu Asn Ser Ser Thr Phe Leu Glu Cys Ser Pro Lys Ser Gln Arg Ala 590 595 600 CTG GTC TAT TGG CAA TTC CAG AGG CGA AAT GAA GAG CGA AAA GAA GAG 1875 Leu Val Tyr Trp Gln Phe Gln Arg Arg Asn Glu Glu Arg Lys Glu Glu 605 610 615 620 ATC AGA GTG GAT GAT CAT ATC ATC AGG ACA GAT CAA GGC CTT CTG CTA 1923 Ile Arg Val Asp Asp His Ile Ile Arg Thr Asp Gln Gly Leu Leu Leu 625 630 635 CGT AGT CTA CAA CAG AAG GAT TCA GGC AAT TAC CTC TGC CAT GCG GTG 1971 Arg Ser Leu Gln Gln Lys Asp Ser Gly Asn Tyr Leu Cys His Ala Val 640 645 650 GAA CAT GGG TTC ATA CAA ACT CTT CTT AAG GTA ACC CTG GAA GTC ATT 2019 Glu His Gly Phe Ile Gln Thr Leu Leu Lys Val Thr Leu Glu Val Ile 655 660 665 GAC ACA GAG CAT TTG GAA GAA CTT CTT CAT AAA GAT GAT GAT GGA GAT 2067 Asp Thr Glu His Leu Glu Glu Leu Leu His Lys Asp Asp Asp Gly Asp 670 675 680 GGC TCT AAG ACC AAA GAA ATG TCC AAT AGC ATG ACA CCT AGC CAG AAG 2115 Gly Ser Lys Thr Lys Glu Met Ser Asn Ser Met Thr Pro Ser Gln Lys 685 690 695 700 GTC TGG TAC AGA GAC TTC ATG CAG CTC ATC AAC CAC CCC AAT CTC AAC 2163 Val Trp Tyr Arg Asp Phe Met Gln Leu Ile Asn His Pro Asn Leu Asn 705 710 715 ACG ATG GAT GAG TTC TGT GAA CAA GTT TGG AAA AGG GAC CGA AAA CAA 2211 Thr Met Asp Glu Phe Cys Glu Gln Val Trp Lys Arg Asp Arg Lys Gln 720 725 730 CGT CGG CAA AGG CCA GGA CAT ACC CCA GGG AAC AGT AAC AAA TGG AAG 2259 Arg Arg Gln Arg Pro Gly His Thr Pro Gly Asn Ser Asn Lys Trp Lys 735 740 745 CAC TTA CAA GAA AAT AAG AAA GGT AGA AAC AGG AGG ACC CAC GAA TTT 2307 His Leu Gln Glu Asn Lys Lys Gly Arg Asn Arg Arg Thr His Glu Phe 750 755 760 GAG AGG GCA CCC AGG AGT GTC TGAGCTGCAT TACCTCTAGA AACCTCAAAC 2358 Glu Arg Ala Pro Arg Ser Val 765 770 AAGTAGAAAC TTGCCTAGAC AATAACTGGA AAAACAAATG CAATATACAT GAACTTTTTT 2418 CATGGCATTA TGTGGATGTT TACAATGGTG GGAAATTCAG CTGAGTTCCA CCAATTATAA 2478 ATTAAATCCA TGAGTAACTT TCCTAATAGG CTTTTTTTTC CTAATACCAC CGGGTTAAAA 2538 GTAAGAGACA GCTGAACCCT CGTGGAGCCA TTCATACAGG TCCCTATTTA AGGAACGGAA 2598 TTC 2601 771 amino acids amino acid linear protein 54 Met Gly Trp Leu Thr Arg Ile Val Cys Leu Phe Trp Gly Val Leu Leu 1 5 10 15 Thr Ala Arg Ala Asn Tyr Gln Asn Gly Lys Asn Asn Val Pro Arg Leu 20 25 30 Lys Leu Ser Tyr Lys Glu Met Leu Glu Ser Asn Asn Val Ile Thr Phe 35 40 45 Asn Gly Leu Ala Asn Ser Ser Ser Tyr His Thr Phe Leu Leu Asp Glu 50 55 60 Glu Arg Ser Arg Leu Tyr Val Gly Ala Lys Asp His Ile Phe Ser Phe 65 70 75 80 Asp Leu Val Asn Ile Lys Asp Phe Gln Lys Ile Val Trp Pro Val Ser 85 90 95 Tyr Thr Arg Arg Asp Glu Cys Lys Trp Ala Gly Lys Asp Ile Leu Lys 100 105 110 Glu Cys Ala Asn Phe Ile Lys Val Leu Lys Ala Tyr Asn Gln Thr His 115 120 125 Leu Tyr Ala Cys Gly Thr Gly Ala Phe His Pro Ile Cys Thr Tyr Ile 130 135 140 Glu Ile Gly His His Pro Glu Asp Asn Ile Phe Lys Leu Glu Asn Ser 145 150 155 160 His Phe Glu Asn Gly Arg Gly Lys Ser Pro Tyr Asp Pro Lys Leu Leu 165 170 175 Thr Ala Ser Leu Leu Ile Asp Gly Glu Leu Tyr Ser Gly Thr Ala Ala 180 185 190 Asp Phe Met Gly Arg Asp Phe Ala Ile Phe Arg Thr Leu Gly His His 195 200 205 His Pro Ile Arg Thr Glu Gln His Asp Ser Arg Trp Leu Asn Asp Pro 210 215 220 Lys Phe Ile Ser Ala His Leu Ile Ser Glu Ser Asp Asn Pro Glu Asp 225 230 235 240 Asp Lys Val Tyr Phe Phe Phe Arg Glu Asn Ala Ile Asp Gly Glu His 245 250 255 Ser Gly Lys Ala Thr His Ala Arg Ile Gly Gln Ile Cys Lys Asn Asp 260 265 270 Phe Gly Gly His Arg Ser Leu Val Asn Lys Trp Thr Thr Phe Leu Lys 275 280 285 Ala Arg Leu Ile Cys Ser Val Pro Gly Pro Asn Gly Ile Asp Thr His 290 295 300 Phe Asp Glu Leu Gln Asp Val Phe Leu Met Asn Phe Lys Asp Pro Lys 305 310 315 320 Asn Pro Val Val Tyr Gly Val Phe Thr Thr Ser Ser Asn Ile Phe Lys 325 330 335 Gly Ser Ala Val Cys Met Tyr Ser Met Ser Asp Val Arg Arg Val Phe 340 345 350 Leu Gly Pro Tyr Ala His Arg Asp Gly Pro Asn Tyr Gln Trp Val Pro 355 360 365 Tyr Gln Gly Arg Val Pro Tyr Pro Arg Pro Gly Thr Cys Pro Ser Lys 370 375 380 Thr Phe Gly Gly Phe Asp Ser Thr Lys Asp Leu Pro Asp Asp Val Ile 385 390 395 400 Thr Phe Ala Arg Ser His Pro Ala Met Tyr Asn Pro Val Phe Pro Met 405 410 415 Asn Asn Arg Pro Ile Val Ile Lys Thr Asp Val Asn Tyr Gln Phe Thr 420 425 430 Gln Ile Val Val Asp Arg Val Asp Ala Glu Asp Gly Gln Tyr Asp Val 435 440 445 Met Phe Ile Gly Thr Asp Val Gly Thr Val Leu Lys Val Val Ser Ile 450 455 460 Pro Lys Glu Thr Trp Tyr Asp Leu Glu Glu Val Leu Leu Glu Glu Met 465 470 475 480 Thr Val Phe Arg Glu Pro Thr Ala Ile Ser Ala Met Glu Leu Ser Thr 485 490 495 Lys Gln Gln Gln Leu Tyr Ile Gly Ser Thr Ala Gly Val Ala Gln Leu 500 505 510 Pro Leu His Arg Cys Asp Ile Tyr Gly Lys Ala Cys Ala Glu Cys Cys 515 520 525 Leu Ala Arg Asp Pro Tyr Cys Ala Trp Asp Gly Ser Ala Cys Ser Arg 530 535 540 Tyr Phe Pro Thr Ala Lys Arg Arg Thr Arg Arg Gln Asp Ile Arg Asn 545 550 555 560 Gly Asp Pro Leu Thr His Cys Ser Asp Leu His His Asp Asn His His 565 570 575 Gly His Ser Pro Glu Glu Arg Ile Ile Tyr Gly Val Glu Asn Ser Ser 580 585 590 Thr Phe Leu Glu Cys Ser Pro Lys Ser Gln Arg Ala Leu Val Tyr Trp 595 600 605 Gln Phe Gln Arg Arg Asn Glu Glu Arg Lys Glu Glu Ile Arg Val Asp 610 615 620 Asp His Ile Ile Arg Thr Asp Gln Gly Leu Leu Leu Arg Ser Leu Gln 625 630 635 640 Gln Lys Asp Ser Gly Asn Tyr Leu Cys His Ala Val Glu His Gly Phe 645 650 655 Ile Gln Thr Leu Leu Lys Val Thr Leu Glu Val Ile Asp Thr Glu His 660 665 670 Leu Glu Glu Leu Leu His Lys Asp Asp Asp Gly Asp Gly Ser Lys Thr 675 680 685 Lys Glu Met Ser Asn Ser Met Thr Pro Ser Gln Lys Val Trp Tyr Arg 690 695 700 Asp Phe Met Gln Leu Ile Asn His Pro Asn Leu Asn Thr Met Asp Glu 705 710 715 720 Phe Cys Glu Gln Val Trp Lys Arg Asp Arg Lys Gln Arg Arg Gln Arg 725 730 735 Pro Gly His Thr Pro Gly Asn Ser Asn Lys Trp Lys His Leu Gln Glu 740 745 750 Asn Lys Lys Gly Arg Asn Arg Arg Thr His Glu Phe Glu Arg Ala Pro 755 760 765 Arg Ser Val 770 1332 base pairs nucleic acid double linear cDNA CDS 7..1329 55 GGAATA ATG ATG GTA TTA TTA CAT GCT GTA TAC TCT ATA GTC TTT GTA 48 Met Met Val Leu Leu His Ala Val Tyr Ser Ile Val Phe Val 1 5 10 GAT GTT ATA ATC ATA AAA GTA CAG AGG TAT ATC AAC GAT ATT CTA ACT 96 Asp Val Ile Ile Ile Lys Val Gln Arg Tyr Ile Asn Asp Ile Leu Thr 15 20 25 30 CTT GAC ATT TTT TAT TTA TTT AAA ATG ATA CCT TTG TTA TTT ATT TTA 144 Leu Asp Ile Phe Tyr Leu Phe Lys Met Ile Pro Leu Leu Phe Ile Leu 35 40 45 TTC TAT TTT GCT AAC GGT ATC GAA TGG CAT AAG TTT GAA ACG AGT GAA 192 Phe Tyr Phe Ala Asn Gly Ile Glu Trp His Lys Phe Glu Thr Ser Glu 50 55 60 GAA ATA ATT TCT ACT TAC TTA TTA GAC GAC GTA TTA TAC ACG GGT GTT 240 Glu Ile Ile Ser Thr Tyr Leu Leu Asp Asp Val Leu Tyr Thr Gly Val 65 70 75 AAT GGG GCG GTA TAC ACA TTT TCA AAT AAT AAA CTA AAC AAA ACT GGT 288 Asn Gly Ala Val Tyr Thr Phe Ser Asn Asn Lys Leu Asn Lys Thr Gly 80 85 90 TTA ACT AAT AAT AAT TAT ATA ACA ACA TCT ATA AAA GTA GAG GAT GCG 336 Leu Thr Asn Asn Asn Tyr Ile Thr Thr Ser Ile Lys Val Glu Asp Ala 95 100 105 110 GAT AAG GAT ACA TTA GTA TGC GGA ACC AAT AAC GGA AAT CCC AAA TGT 384 Asp Lys Asp Thr Leu Val Cys Gly Thr Asn Asn Gly Asn Pro Lys Cys 115 120 125 TGG AAA ATA GAC GGT TCA GAC GAC CCA AAA CAT AGA GGT AGA GGA TAC 432 Trp Lys Ile Asp Gly Ser Asp Asp Pro Lys His Arg Gly Arg Gly Tyr 130 135 140 GCT CCT TAT CAA AAT AGC AAA GTA ACG ATA ATC AGT CAC AAC GGA TGT 480 Ala Pro Tyr Gln Asn Ser Lys Val Thr Ile Ile Ser His Asn Gly Cys 145 150 155 GTA CTA TCT GAC ATA AAC ATA TCA AAA GAA GGA ATT AAA CGA TGG AGA 528 Val Leu Ser Asp Ile Asn Ile Ser Lys Glu Gly Ile Lys Arg Trp Arg 160 165 170 AGA TTT GAC GGA CCA TGT GGT TAT GAT TTA TAC ACG GCG GAT AAC GTA 576 Arg Phe Asp Gly Pro Cys Gly Tyr Asp Leu Tyr Thr Ala Asp Asn Val 175 180 185 190 ATT CCA AAA GAT GGT TTA CGA GGA GCA TTC GTC GAT AAA GAT GGT ACT 624 Ile Pro Lys Asp Gly Leu Arg Gly Ala Phe Val Asp Lys Asp Gly Thr 195 200 205 TAT GAC AAA GTT TAC ATT CTT TTC ACT GAT ACT ATC GGC TCA AAG AGA 672 Tyr Asp Lys Val Tyr Ile Leu Phe Thr Asp Thr Ile Gly Ser Lys Arg 210 215 220 ATT GTC AAA ATT CCG TAT ATA GCA CAA ATG TGC CTA AAC GAC GAA GGT 720 Ile Val Lys Ile Pro Tyr Ile Ala Gln Met Cys Leu Asn Asp Glu Gly 225 230 235 GGT CCA TCA TCA TTG TCT AGT CAT AGA TGG TCG ACG TTT CTC AAA GTC 768 Gly Pro Ser Ser Leu Ser Ser His Arg Trp Ser Thr Phe Leu Lys Val 240 245 250 GAA TTA GAA TGT GAT ATC GAC GGA AGA AGT TAT AGA CAA ATT ATT CAT 816 Glu Leu Glu Cys Asp Ile Asp Gly Arg Ser Tyr Arg Gln Ile Ile His 255 260 265 270 TCT AGA ACT ATA AAA ACA GAT AAT GAT ACG ATA CTA TAT GTA TTC TTC 864 Ser Arg Thr Ile Lys Thr Asp Asn Asp Thr Ile Leu Tyr Val Phe Phe 275 280 285 GAT AGT CCT TAT TCC AAG TCC GCA TTA TGT ACC TAT TCT ATG AAT ACC 912 Asp Ser Pro Tyr Ser Lys Ser Ala Leu Cys Thr Tyr Ser Met Asn Thr 290 295 300 ATT AAA CAA TCT TTT TCT ACG TCA AAA TTG GAA GGA TAT ACA AAG CAA 960 Ile Lys Gln Ser Phe Ser Thr Ser Lys Leu Glu Gly Tyr Thr Lys Gln 305 310 315 TTG CCG TCG CCA GCC TCT GGT ATA TGT CTA CCA GCT GGA AAA GTT GTT 1008 Leu Pro Ser Pro Ala Ser Gly Ile Cys Leu Pro Ala Gly Lys Val Val 320 325 330 CCA CAT ACC ACG TTT GAA GTC ATA GAA AAA TAT AAT GTA CTA GAT GAT 1056 Pro His Thr Thr Phe Glu Val Ile Glu Lys Tyr Asn Val Leu Asp Asp 335 340 345 350 ATT ATA AAG CCT TTA TCT AAC CAA CCT ATC TTC GAA GGA CCG TCT GGT 1104 Ile Ile Lys Pro Leu Ser Asn Gln Pro Ile Phe Glu Gly Pro Ser Gly 355 360 365 GTT AAA TGG TTC GAT ATA AAG GAG AAG GAA AAT GAA CAT CGG GAA TAT 1152 Val Lys Trp Phe Asp Ile Lys Glu Lys Glu Asn Glu His Arg Glu Tyr 370 375 380 AGA ATA TAC TTC ATA AAA GAA AAT TCT ATA TAT TCG TTC GAT ACA AAA 1200 Arg Ile Tyr Phe Ile Lys Glu Asn Ser Ile Tyr Ser Phe Asp Thr Lys 385 390 395 TCT AAA CAA ACT CGT AGC TCG CAA GTC GAT GCG CGA CTA TTT TCA GTA 1248 Ser Lys Gln Thr Arg Ser Ser Gln Val Asp Ala Arg Leu Phe Ser Val 400 405 410 ATG GTA ACT TCG AAA CCG TTA TTT ATA GCA GAT ATA GGG ATA GGA GTA 1296 Met Val Thr Ser Lys Pro Leu Phe Ile Ala Asp Ile Gly Ile Gly Val 415 420 425 430 GGA ATG CCA CAA ATG AAA AAA ATA CTT AAA ATG TAA 1332 Gly Met Pro Gln Met Lys Lys Ile Leu Lys Met 435 440 441 amino acids amino acid linear protein 56 Met Met Val Leu Leu His Ala Val Tyr Ser Ile Val Phe Val Asp Val 1 5 10 15 Ile Ile Ile Lys Val Gln Arg Tyr Ile Asn Asp Ile Leu Thr Leu Asp 20 25 30 Ile Phe Tyr Leu Phe Lys Met Ile Pro Leu Leu Phe Ile Leu Phe Tyr 35 40 45 Phe Ala Asn Gly Ile Glu Trp His Lys Phe Glu Thr Ser Glu Glu Ile 50 55 60 Ile Ser Thr Tyr Leu Leu Asp Asp Val Leu Tyr Thr Gly Val Asn Gly 65 70 75 80 Ala Val Tyr Thr Phe Ser Asn Asn Lys Leu Asn Lys Thr Gly Leu Thr 85 90 95 Asn Asn Asn Tyr Ile Thr Thr Ser Ile Lys Val Glu Asp Ala Asp Lys 100 105 110 Asp Thr Leu Val Cys Gly Thr Asn Asn Gly Asn Pro Lys Cys Trp Lys 115 120 125 Ile Asp Gly Ser Asp Asp Pro Lys His Arg Gly Arg Gly Tyr Ala Pro 130 135 140 Tyr Gln Asn Ser Lys Val Thr Ile Ile Ser His Asn Gly Cys Val Leu 145 150 155 160 Ser Asp Ile Asn Ile Ser Lys Glu Gly Ile Lys Arg Trp Arg Arg Phe 165 170 175 Asp Gly Pro Cys Gly Tyr Asp Leu Tyr Thr Ala Asp Asn Val Ile Pro 180 185 190 Lys Asp Gly Leu Arg Gly Ala Phe Val Asp Lys Asp Gly Thr Tyr Asp 195 200 205 Lys Val Tyr Ile Leu Phe Thr Asp Thr Ile Gly Ser Lys Arg Ile Val 210 215 220 Lys Ile Pro Tyr Ile Ala Gln Met Cys Leu Asn Asp Glu Gly Gly Pro 225 230 235 240 Ser Ser Leu Ser Ser His Arg Trp Ser Thr Phe Leu Lys Val Glu Leu 245 250 255 Glu Cys Asp Ile Asp Gly Arg Ser Tyr Arg Gln Ile Ile His Ser Arg 260 265 270 Thr Ile Lys Thr Asp Asn Asp Thr Ile Leu Tyr Val Phe Phe Asp Ser 275 280 285 Pro Tyr Ser Lys Ser Ala Leu Cys Thr Tyr Ser Met Asn Thr Ile Lys 290 295 300 Gln Ser Phe Ser Thr Ser Lys Leu Glu Gly Tyr Thr Lys Gln Leu Pro 305 310 315 320 Ser Pro Ala Ser Gly Ile Cys Leu Pro Ala Gly Lys Val Val Pro His 325 330 335 Thr Thr Phe Glu Val Ile Glu Lys Tyr Asn Val Leu Asp Asp Ile Ile 340 345 350 Lys Pro Leu Ser Asn Gln Pro Ile Phe Glu Gly Pro Ser Gly Val Lys 355 360 365 Trp Phe Asp Ile Lys Glu Lys Glu Asn Glu His Arg Glu Tyr Arg Ile 370 375 380 Tyr Phe Ile Lys Glu Asn Ser Ile Tyr Ser Phe Asp Thr Lys Ser Lys 385 390 395 400 Gln Thr Arg Ser Ser Gln Val Asp Ala Arg Leu Phe Ser Val Met Val 405 410 415 Thr Ser Lys Pro Leu Phe Ile Ala Asp Ile Gly Ile Gly Val Gly Met 420 425 430 Pro Gln Met Lys Lys Ile Leu Lys Met 435 440 2854 base pairs nucleic acid double linear cDNA CDS 451..2640 57 ATTCCACCTC CCGCTGACCG CCTACGCCGC GACGATCTTT CCTCTCGCCA GGCGAAAACT 60 ACGACGTGTC AACAACATTT TTGTTTTTTC TGCTTCCGTG TTTTCATGTT CCGTGAAACC 120 GCTTCTCGCA TTACCACTCT TCCGTTTCCC AGTGTTTGTT TTCTCCGTTT CTTTCATCGT 180 GGATGTTTTG TTTTGGTGTA GCGAGTGACG AGCTTATGTC ATTAAACGTA CATCCAATCT 240 GTCGGTATAT TGGTGTGTGA TATTTTACTA TTATATATTT AGCCATCACT TGAAAGCCGT 300 GAAAAATTTT TGAAAGTGGA GAGGAAAAAG AAAAGGCGCA GAAGGCTTTT TAAGCTTCAT 360 GGATATGTGC TCTACGCTTC AACTACTGTC GCAGAATCAT CTTCCGGGAA AGGAAATTTC 420 GCCTGAAATG GTGCCGCGGC CGCACTGAAC ATG CGG GCG GCG CTG GTG GCC GTC 474 Met Arg Ala Ala Leu Val Ala Val 1 5 GCG GCG CTG CTT TGG GTG GCG CTG CAC GCC GCC GCA TGG GTC AAC GAC 522 Ala Ala Leu Leu Trp Val Ala Leu His Ala Ala Ala Trp Val Asn Asp 10 15 20 GTC AGC CCC AAG ATG TAC GTC CAG TTC GGT GAG GAA CGG GTG CAA CGC 570 Val Ser Pro Lys Met Tyr Val Gln Phe Gly Glu Glu Arg Val Gln Arg 25 30 35 40 TTC CTG GGC AAT GAA TCG CAC AAA GAC CAC TTC AAG CTG CTG GAG AAG 618 Phe Leu Gly Asn Glu Ser His Lys Asp His Phe Lys Leu Leu Glu Lys 45 50 55 GAC CAC AAC TCG CTC CTC GTA GGA GCT AGG AAC ATC GTC TAC AAT ATC 666 Asp His Asn Ser Leu Leu Val Gly Ala Arg Asn Ile Val Tyr Asn Ile 60 65 70 AGC CTT CGA GAC CTC ACA GAA TTC ACC GAG CAG AGG ATC GAG TGG CAC 714 Ser Leu Arg Asp Leu Thr Glu Phe Thr Glu Gln Arg Ile Glu Trp His 75 80 85 TCG TCA GGT GCC CAT CGC GAG CTC TGC TAC CTC AAG GGG AAG TCA GAG 762 Ser Ser Gly Ala His Arg Glu Leu Cys Tyr Leu Lys Gly Lys Ser Glu 90 95 100 GAC GAC TGC CAG AAC TAC ATC CGA GTC CTG GCG AAA ATT GAC GAT GAC 810 Asp Asp Cys Gln Asn Tyr Ile Arg Val Leu Ala Lys Ile Asp Asp Asp 105 110 115 120 CGC GTA CTC ATC TGC GGT ACG AAC GCC TAT AAG CCA CTA TGT CGG CAC 858 Arg Val Leu Ile Cys Gly Thr Asn Ala Tyr Lys Pro Leu Cys Arg His 125 130 135 TAC GCC CTC AAG GAT GGA GAT TAT GTT GTA GAG AAA GAA TAT GAG GGA 906 Tyr Ala Leu Lys Asp Gly Asp Tyr Val Val Glu Lys Glu Tyr Glu Gly 140 145 150 AGA GGA TTG TGC CCA TTT GAC CCT GAC CAC AAC AGC ACT GCA ATA TAC 954 Arg Gly Leu Cys Pro Phe Asp Pro Asp His Asn Ser Thr Ala Ile Tyr 155 160 165 AGT GAG GGA CAA TTG TAC TCA GCA ACA GTG GCA GAC TTC TCT GGA ACT 1002 Ser Glu Gly Gln Leu Tyr Ser Ala Thr Val Ala Asp Phe Ser Gly Thr 170 175 180 GAC CCT CTC ATA TAC CGC GGC CCT CTA AGA ACA GAG AGA TCT GAC CTC 1050 Asp Pro Leu Ile Tyr Arg Gly Pro Leu Arg Thr Glu Arg Ser Asp Leu 185 190 195 200 AAA CAA TTA AAT GCT CCT AAC TTT GTC AAC ACA ATG GAG TAC AAT GAT 1098 Lys Gln Leu Asn Ala Pro Asn Phe Val Asn Thr Met Glu Tyr Asn Asp 205 210 215 TTT ATA TTC TTC TTC TTC CGA GAG ACT GCT GTT GAG TAC ATC AAC TGC 1146 Phe Ile Phe Phe Phe Phe Arg Glu Thr Ala Val Glu Tyr Ile Asn Cys 220 225 230 GGA AAG GCT ATC TAT TCA AGA GTT GCC AGA GTC TGT AAA CAT GAC AAG 1194 Gly Lys Ala Ile Tyr Ser Arg Val Ala Arg Val Cys Lys His Asp Lys 235 240 245 GGC GGC CCT CAT CAG GGT GGT GAC AGA TGG ACT TCT TTT TTG AAA TCA 1242 Gly Gly Pro His Gln Gly Gly Asp Arg Trp Thr Ser Phe Leu Lys Ser 250 255 260 CGT CTG AAC TGT TCC GTC CCT GGA GAT TAT CCA TTT TAC TTC AAT GAA 1290 Arg Leu Asn Cys Ser Val Pro Gly Asp Tyr Pro Phe Tyr Phe Asn Glu 265 270 275 280 ATT CAG TCA ACA AGT GAC ATC ATT GAA GGA AAT TAT GGT GGT CAA GTG 1338 Ile Gln Ser Thr Ser Asp Ile Ile Glu Gly Asn Tyr Gly Gly Gln Val 285 290 295 GAG AAA CTC ATC TAC GGT GTC TTC ACG ACA CCA GTG AAC TCT ATT GGT 1386 Glu Lys Leu Ile Tyr Gly Val Phe Thr Thr Pro Val Asn Ser Ile Gly 300 305 310 GGC TCT GCT GTT TGT GCC TTC AGT ATG AAG TCA ATA CTT GAG TCA TTT 1434 Gly Ser Ala Val Cys Ala Phe Ser Met Lys Ser Ile Leu Glu Ser Phe 315 320 325 GAT GGT CCA TTT AAA GAG CAG GAA ACG ATG AAC TCA AAC TGG TTG GCA 1482 Asp Gly Pro Phe Lys Glu Gln Glu Thr Met Asn Ser Asn Trp Leu Ala 330 335 340 GTG CCA AGC CTT AAA GTG CCA GAA CCA AGG CCT GGA CAA TGT GTG AAT 1530 Val Pro Ser Leu Lys Val Pro Glu Pro Arg Pro Gly Gln Cys Val Asn 345 350 355 360 GAC AGT CGT ACA CTT CCT GAT GTG TCT GTC AAT TTT GTA AAG TCA CAT 1578 Asp Ser Arg Thr Leu Pro Asp Val Ser Val Asn Phe Val Lys Ser His 365 370 375 ACA CTG ATG GAT GAG GCC GTG CCA GCA TTT TTT ACT CGG CCA ATT CTC 1626 Thr Leu Met Asp Glu Ala Val Pro Ala Phe Phe Thr Arg Pro Ile Leu 380 385 390 ATT CGG ATC AGC TTA CAG TAC AGA TTT ACA AAA ATA GCT GTT GAT CAA 1674 Ile Arg Ile Ser Leu Gln Tyr Arg Phe Thr Lys Ile Ala Val Asp Gln 395 400 405 CAA GTC CGA ACA CCA GAT GGG AAA GCG TAT GAT GTC CTG TTT ATA GGA 1722 Gln Val Arg Thr Pro Asp Gly Lys Ala Tyr Asp Val Leu Phe Ile Gly 410 415 420 ACT GAT GAT GGC AAA GTG ATA AAA GCT TTG AAC TCT GCC TCC TTT GAT 1770 Thr Asp Asp Gly Lys Val Ile Lys Ala Leu Asn Ser Ala Ser Phe Asp 425 430 435 440 TCA TCT GAT ACT GTA GAT AGT GTT GTA ATA GAA GAA CTG CAA GTG TTG 1818 Ser Ser Asp Thr Val Asp Ser Val Val Ile Glu Glu Leu Gln Val Leu 445 450 455 CCA CCT GGA GTA CCT GTT AAG AAC CTG TAT GTG GTG CGA ATG GAT GGG 1866 Pro Pro Gly Val Pro Val Lys Asn Leu Tyr Val Val Arg Met Asp Gly 460 465 470 GAT GAT AGC AAG CTG GTG GTT GTG TCT GAT GAT GAG ATT CTG GCA ATT 1914 Asp Asp Ser Lys Leu Val Val Val Ser Asp Asp Glu Ile Leu Ala Ile 475 480 485 AAG CTT CAT CGT TGT GGC TCA GAT AAA ATA ACA AAT TGT CGA GAA TGT 1962 Lys Leu His Arg Cys Gly Ser Asp Lys Ile Thr Asn Cys Arg Glu Cys 490 495 500 GTG TCC TTG CAA GAT CCT TAC TGT GCA TGG GAC AAT GTA GAA TTA AAA 2010 Val Ser Leu Gln Asp Pro Tyr Cys Ala Trp Asp Asn Val Glu Leu Lys 505 510 515 520 TGT ACA GCT GTA GGT TCA CCA GAC TGG AGT GCT GGA AAA AGA CGC TTT 2058 Cys Thr Ala Val Gly Ser Pro Asp Trp Ser Ala Gly Lys Arg Arg Phe 525 530 535 ATT CAG AAC ATT TCA CTC GGT GAA CAT AAA GCT TGT GGT GGA CGT CCA 2106 Ile Gln Asn Ile Ser Leu Gly Glu His Lys Ala Cys Gly Gly Arg Pro 540 545 550 CAA ACA GAA ATC GTT GCT TCT CCT GTA CCA ACT CAG CCG ACG ACA AAA 2154 Gln Thr Glu Ile Val Ala Ser Pro Val Pro Thr Gln Pro Thr Thr Lys 555 560 565 TCT AGT GGC GAT CCC GTT CAT TCA ATC CAC CAG GCT GAA TTT GAA CCT 2202 Ser Ser Gly Asp Pro Val His Ser Ile His Gln Ala Glu Phe Glu Pro 570 575 580 GAA ATT GAC AAC GAG ATT GTT ATT GGA GTA GAT GAC AGC AAC GTC ATT 2250 Glu Ile Asp Asn Glu Ile Val Ile Gly Val Asp Asp Ser Asn Val Ile 585 590 595 600 CCT AAT ACC CTG GCT GAA ATA AAT CAT GCA GGT TCA AAG CTG CCT TCC 2298 Pro Asn Thr Leu Ala Glu Ile Asn His Ala Gly Ser Lys Leu Pro Ser 605 610 615 TCC CAG GAA AAG TTG CCT ATT TAT ACA GCG GAG ACT CTG ACT ATT GCT 2346 Ser Gln Glu Lys Leu Pro Ile Tyr Thr Ala Glu Thr Leu Thr Ile Ala 620 625 630 ATA GTT ACA TCA TGC CTT GGA GCT CTA GTT GTT GGC TTC ATC TCT GGA 2394 Ile Val Thr Ser Cys Leu Gly Ala Leu Val Val Gly Phe Ile Ser Gly 635 640 645 TTT CTT TTT TCT CGG CGA TGC AGG GGA GAG GAT TAC ACA GAC ATG CCT 2442 Phe Leu Phe Ser Arg Arg Cys Arg Gly Glu Asp Tyr Thr Asp Met Pro 650 655 660 TTT CCA GAT CAA CGC CAT CAG CTA AAT AGG CTC ACT GAG GCT GGT CTG 2490 Phe Pro Asp Gln Arg His Gln Leu Asn Arg Leu Thr Glu Ala Gly Leu 665 670 675 680 AAT GCA GAC TCA CCC TAT CTT CCA CCC TGT GCC AAT AAC AAG GCA GCC 2538 Asn Ala Asp Ser Pro Tyr Leu Pro Pro Cys Ala Asn Asn Lys Ala Ala 685 690 695 ATA AAT CTT GTG CTC AAT GTC CCA CCA AAG AAT GCA AAT GGA AAA AAT 2586 Ile Asn Leu Val Leu Asn Val Pro Pro Lys Asn Ala Asn Gly Lys Asn 700 705 710 GCC AAC TCT TCA GCT GAA AAC AAA CCA ATA CAG AAA GTA AAA AAG ACA 2634 Ala Asn Ser Ser Ala Glu Asn Lys Pro Ile Gln Lys Val Lys Lys Thr 715 720 725 TAC ATT TAGCAGAAAT CTTTGGTATC TGTTTTGGTG CAGACCCATG CCACTAGAGT 2690 Tyr Ile 730 AACCAAGACT CTATTGAGAA ATGTCCTCAA GAAAGTTAAA AAGATGTAGA CTTCTGTAAT 2750 CGAGAGCACC ACTTTCCATA GTAATACAGA ACAATGTGAA ATAAATACTA CAGAAGAAGT 2810 CTTTGTTACA CAAAAAAGTG TATAGTGATC TGTGATCAGT TTCG 2854 730 amino acids amino acid linear protein 58 Met Arg Ala Ala Leu Val Ala Val Ala Ala Leu Leu Trp Val Ala Leu 1 5 10 15 His Ala Ala Ala Trp Val Asn Asp Val Ser Pro Lys Met Tyr Val Gln 20 25 30 Phe Gly Glu Glu Arg Val Gln Arg Phe Leu Gly Asn Glu Ser His Lys 35 40 45 Asp His Phe Lys Leu Leu Glu Lys Asp His Asn Ser Leu Leu Val Gly 50 55 60 Ala Arg Asn Ile Val Tyr Asn Ile Ser Leu Arg Asp Leu Thr Glu Phe 65 70 75 80 Thr Glu Gln Arg Ile Glu Trp His Ser Ser Gly Ala His Arg Glu Leu 85 90 95 Cys Tyr Leu Lys Gly Lys Ser Glu Asp Asp Cys Gln Asn Tyr Ile Arg 100 105 110 Val Leu Ala Lys Ile Asp Asp Asp Arg Val Leu Ile Cys Gly Thr Asn 115 120 125 Ala Tyr Lys Pro Leu Cys Arg His Tyr Ala Leu Lys Asp Gly Asp Tyr 130 135 140 Val Val Glu Lys Glu Tyr Glu Gly Arg Gly Leu Cys Pro Phe Asp Pro 145 150 155 160 Asp His Asn Ser Thr Ala Ile Tyr Ser Glu Gly Gln Leu Tyr Ser Ala 165 170 175 Thr Val Ala Asp Phe Ser Gly Thr Asp Pro Leu Ile Tyr Arg Gly Pro 180 185 190 Leu Arg Thr Glu Arg Ser Asp Leu Lys Gln Leu Asn Ala Pro Asn Phe 195 200 205 Val Asn Thr Met Glu Tyr Asn Asp Phe Ile Phe Phe Phe Phe Arg Glu 210 215 220 Thr Ala Val Glu Tyr Ile Asn Cys Gly Lys Ala Ile Tyr Ser Arg Val 225 230 235 240 Ala Arg Val Cys Lys His Asp Lys Gly Gly Pro His Gln Gly Gly Asp 245 250 255 Arg Trp Thr Ser Phe Leu Lys Ser Arg Leu Asn Cys Ser Val Pro Gly 260 265 270 Asp Tyr Pro Phe Tyr Phe Asn Glu Ile Gln Ser Thr Ser Asp Ile Ile 275 280 285 Glu Gly Asn Tyr Gly Gly Gln Val Glu Lys Leu Ile Tyr Gly Val Phe 290 295 300 Thr Thr Pro Val Asn Ser Ile Gly Gly Ser Ala Val Cys Ala Phe Ser 305 310 315 320 Met Lys Ser Ile Leu Glu Ser Phe Asp Gly Pro Phe Lys Glu Gln Glu 325 330 335 Thr Met Asn Ser Asn Trp Leu Ala Val Pro Ser Leu Lys Val Pro Glu 340 345 350 Pro Arg Pro Gly Gln Cys Val Asn Asp Ser Arg Thr Leu Pro Asp Val 355 360 365 Ser Val Asn Phe Val Lys Ser His Thr Leu Met Asp Glu Ala Val Pro 370 375 380 Ala Phe Phe Thr Arg Pro Ile Leu Ile Arg Ile Ser Leu Gln Tyr Arg 385 390 395 400 Phe Thr Lys Ile Ala Val Asp Gln Gln Val Arg Thr Pro Asp Gly Lys 405 410 415 Ala Tyr Asp Val Leu Phe Ile Gly Thr Asp Asp Gly Lys Val Ile Lys 420 425 430 Ala Leu Asn Ser Ala Ser Phe Asp Ser Ser Asp Thr Val Asp Ser Val 435 440 445 Val Ile Glu Glu Leu Gln Val Leu Pro Pro Gly Val Pro Val Lys Asn 450 455 460 Leu Tyr Val Val Arg Met Asp Gly Asp Asp Ser Lys Leu Val Val Val 465 470 475 480 Ser Asp Asp Glu Ile Leu Ala Ile Lys Leu His Arg Cys Gly Ser Asp 485 490 495 Lys Ile Thr Asn Cys Arg Glu Cys Val Ser Leu Gln Asp Pro Tyr Cys 500 505 510 Ala Trp Asp Asn Val Glu Leu Lys Cys Thr Ala Val Gly Ser Pro Asp 515 520 525 Trp Ser Ala Gly Lys Arg Arg Phe Ile Gln Asn Ile Ser Leu Gly Glu 530 535 540 His Lys Ala Cys Gly Gly Arg Pro Gln Thr Glu Ile Val Ala Ser Pro 545 550 555 560 Val Pro Thr Gln Pro Thr Thr Lys Ser Ser Gly Asp Pro Val His Ser 565 570 575 Ile His Gln Ala Glu Phe Glu Pro Glu Ile Asp Asn Glu Ile Val Ile 580 585 590 Gly Val Asp Asp Ser Asn Val Ile Pro Asn Thr Leu Ala Glu Ile Asn 595 600 605 His Ala Gly Ser Lys Leu Pro Ser Ser Gln Glu Lys Leu Pro Ile Tyr 610 615 620 Thr Ala Glu Thr Leu Thr Ile Ala Ile Val Thr Ser Cys Leu Gly Ala 625 630 635 640 Leu Val Val Gly Phe Ile Ser Gly Phe Leu Phe Ser Arg Arg Cys Arg 645 650 655 Gly Glu Asp Tyr Thr Asp Met Pro Phe Pro Asp Gln Arg His Gln Leu 660 665 670 Asn Arg Leu Thr Glu Ala Gly Leu Asn Ala Asp Ser Pro Tyr Leu Pro 675 680 685 Pro Cys Ala Asn Asn Lys Ala Ala Ile Asn Leu Val Leu Asn Val Pro 690 695 700 Pro Lys Asn Ala Asn Gly Lys Asn Ala Asn Ser Ser Ala Glu Asn Lys 705 710 715 720 Pro Ile Gln Lys Val Lys Lys Thr Tyr Ile 725 730 3560 base pairs nucleic acid double linear cDNA CDS 1..1953 59 GAG GAT GAT TGT CAG AAT TAC ATC CGC ATC ATG GTG GTG CCA TCG CCG 48 Glu Asp Asp Cys Gln Asn Tyr Ile Arg Ile Met Val Val Pro Ser Pro 1 5 10 15 GGT CGC CTT TTC GTT TGT GGC ACC AAC TCG TTC CGG CCC ATG TGC AAC 96 Gly Arg Leu Phe Val Cys Gly Thr Asn Ser Phe Arg Pro Met Cys Asn 20 25 30 ACG TAT ATC ATT AGT GAC AGC AAC TAC ACG CTG GAG GCC ACG AAG AAC 144 Thr Tyr Ile Ile Ser Asp Ser Asn Tyr Thr Leu Glu Ala Thr Lys Asn 35 40 45 GGA CAG GCG GTG TGC CCC TAC GAT CCA CGT CAC AAC TCC ACC TCT GTG 192 Gly Gln Ala Val Cys Pro Tyr Asp Pro Arg His Asn Ser Thr Ser Val 50 55 60 CTG GCC GAC AAC GAA CTG TAT TCC GGT ACC GTG GCG GAT TTC AGT GGC 240 Leu Ala Asp Asn Glu Leu Tyr Ser Gly Thr Val Ala Asp Phe Ser Gly 65 70 75 80 AGC GAT CCG ATT ATC TAC CGG GAG CCC CTG CAG ACC GAG CAG TAC GAT 288 Ser Asp Pro Ile Ile Tyr Arg Glu Pro Leu Gln Thr Glu Gln Tyr Asp 85 90 95 AGC CTA AGT CTC AAC GCA CCG AAC TTT GTG AGC TCA TTT ACG CAG GGC 336 Ser Leu Ser Leu Asn Ala Pro Asn Phe Val Ser Ser Phe Thr Gln Gly 100 105 110 GAC TTT GTC TAT TTC TTC TTT CGG GAA ACC GCC GTT GAG TTT ATC AAC 384 Asp Phe Val Tyr Phe Phe Phe Arg Glu Thr Ala Val Glu Phe Ile Asn 115 120 125 TGT GGC AAG GCG ATT TAT TCG CGC GTT GCC CGC GTC TGC AAA TGG GAC 432 Cys Gly Lys Ala Ile Tyr Ser Arg Val Ala Arg Val Cys Lys Trp Asp 130 135 140 AAA GGT GGC CCG CAT CGA TTC CGC AAC CGC TGG ACA TCC TTC CTC AAG 480 Lys Gly Gly Pro His Arg Phe Arg Asn Arg Trp Thr Ser Phe Leu Lys 145 150 155 160 TCC CGC CTC AAC TGC TCC ATT CCC GGC GAT TAT CCT TTC TAC TTT AAT 528 Ser Arg Leu Asn Cys Ser Ile Pro Gly Asp Tyr Pro Phe Tyr Phe Asn 165 170 175 GAA ATC CAA TCT GCC AGC AAT CTG GTG GAG GGA CAG TAT GGC TCG ATG 576 Glu Ile Gln Ser Ala Ser Asn Leu Val Glu Gly Gln Tyr Gly Ser Met 180 185 190 AGC TCG AAA CTG ATC TAC GGA GTC TTC AAC ACG CCG AGC AAC TCA ATT 624 Ser Ser Lys Leu Ile Tyr Gly Val Phe Asn Thr Pro Ser Asn Ser Ile 195 200 205 CCC GGC TCA GCG GTT TGT GCC TTT GCC CTC CAG GAC ATT GCC GAT ACG 672 Pro Gly Ser Ala Val Cys Ala Phe Ala Leu Gln Asp Ile Ala Asp Thr 210 215 220 TTT GAG GGT CAG TTC AAG GAG CAG ACT GGC ATC AAC TCC AAC TGG CTG 720 Phe Glu Gly Gln Phe Lys Glu Gln Thr Gly Ile Asn Ser Asn Trp Leu 225 230 235 240 CCA GTG AAC AAC GCC AAG GTA CCC GAT CCT CGA CCC GGT TCC TGT CAC 768 Pro Val Asn Asn Ala Lys Val Pro Asp Pro Arg Pro Gly Ser Cys His 245 250 255 AAC GAT TCG AGA GCG CTT CCG GAT CCC ACA CTG AAC TTC ATC AAA ACA 816 Asn Asp Ser Arg Ala Leu Pro Asp Pro Thr Leu Asn Phe Ile Lys Thr 260 265 270 CAT TCG CTA ATG GAC GAG AAT GTG CCG GCA TTT TTC AGT CAA CCG ATT 864 His Ser Leu Met Asp Glu Asn Val Pro Ala Phe Phe Ser Gln Pro Ile 275 280 285 TTG GTC CGG ACG AGC ACA ATA TAC CGC TTC ACT CAA ATC GCC GTA GAT 912 Leu Val Arg Thr Ser Thr Ile Tyr Arg Phe Thr Gln Ile Ala Val Asp 290 295 300 GCG CAG ATT AAA ACT CCT GGC GGC AAG ACA TAT GAT GTT ATC TTT GTG 960 Ala Gln Ile Lys Thr Pro Gly Gly Lys Thr Tyr Asp Val Ile Phe Val 305 310 315 320 GGC ACA GAT CAT GGA AAG ATT ATT AAG TCA GTG AAT GCT GAA TCT GCC 1008 Gly Thr Asp His Gly Lys Ile Ile Lys Ser Val Asn Ala Glu Ser Ala 325 330 335 GAT TCA GCG GAT AAA GTC ACC TCC GTA GTC ATC GAG GAG ATC GAT GTC 1056 Asp Ser Ala Asp Lys Val Thr Ser Val Val Ile Glu Glu Ile Asp Val 340 345 350 CTG ACC AAG AGT GAA CCC ATA CGC AAT CTG GAG ATA GTC AGA ACC ATG 1104 Leu Thr Lys Ser Glu Pro Ile Arg Asn Leu Glu Ile Val Arg Thr Met 355 360 365 CAG TAC GAT CAA CCC AAA GAT GGC AGC TAC GAC GAT GGT AAA TTA ATC 1152 Gln Tyr Asp Gln Pro Lys Asp Gly Ser Tyr Asp Asp Gly Lys Leu Ile 370 375 380 ATT GTG ACG GAC AGT CAG GTG GTA GCC ATA CAA TTG CAT CGT TGT CAC 1200 Ile Val Thr Asp Ser Gln Val Val Ala Ile Gln Leu His Arg Cys His 385 390 395 400 AAT GAC AAA ATC ACC AGC TGC AGC GAG TGC GTC GCA TTG CAG GAT CCG 1248 Asn Asp Lys Ile Thr Ser Cys Ser Glu Cys Val Ala Leu Gln Asp Pro 405 410 415 TAC TGC GCC TGG GAC AAA ATC GCT GGC AAG TGC CGT TCC CAC GGC GCT 1296 Tyr Cys Ala Trp Asp Lys Ile Ala Gly Lys Cys Arg Ser His Gly Ala 420 425 430 CCC CGA TGG CTA GAG GAG AAC TAT TTC TAC CAG AAT GTG GCC ACT GGC 1344 Pro Arg Trp Leu Glu Glu Asn Tyr Phe Tyr Gln Asn Val Ala Thr Gly 435 440 445 CAG CAT GCG GCC TGC CCC TCA GGC AAA ATC AAT TCA AAG GAT GCC AAC 1392 Gln His Ala Ala Cys Pro Ser Gly Lys Ile Asn Ser Lys Asp Ala Asn 450 455 460 GCT GGG GAG CAG AAG GGC TTC CGC AAC GAC ATG GAC TTA TTG GAT TCG 1440 Ala Gly Glu Gln Lys Gly Phe Arg Asn Asp Met Asp Leu Leu Asp Ser 465 470 475 480 CGA CGC CAG AGC AAG GAT CAG GAA ATA ATC GAC AAT ATT GAT AAG AAC 1488 Arg Arg Gln Ser Lys Asp Gln Glu Ile Ile Asp Asn Ile Asp Lys Asn 485 490 495 TTT GAA GAT ATA ATC AAC GCC CAG TAC ACT GTG GAG ACC CTC GTG ATG 1536 Phe Glu Asp Ile Ile Asn Ala Gln Tyr Thr Val Glu Thr Leu Val Met 500 505 510 GCC GTT CTG GCC GGT TCG ATC TTT TCG CTG CTG GTC GGC TTC TTT ACA 1584 Ala Val Leu Ala Gly Ser Ile Phe Ser Leu Leu Val Gly Phe Phe Thr 515 520 525 GGC TAC TTC TGC GGT CGC CGT TGT CAC AAG GAC GAG GAT GAT AAT CTG 1632 Gly Tyr Phe Cys Gly Arg Arg Cys His Lys Asp Glu Asp Asp Asn Leu 530 535 540 CCG TAT CCG GAT ACG GAG TAC GAG TAC TTC GAG CAG CGA CAG AAT GTC 1680 Pro Tyr Pro Asp Thr Glu Tyr Glu Tyr Phe Glu Gln Arg Gln Asn Val 545 550 555 560 AAT AGC TTC CCC TCG TCC TGT CGC ATC CAG CAG GAG CCC AAG CTG CTG 1728 Asn Ser Phe Pro Ser Ser Cys Arg Ile Gln Gln Glu Pro Lys Leu Leu 565 570 575 CCC CAA GTG GAG GAG GTG ACG TAT GCG GAC GCA GTG CTC CTG CCA CAG 1776 Pro Gln Val Glu Glu Val Thr Tyr Ala Asp Ala Val Leu Leu Pro Gln 580 585 590 CCT CCG CCG CCC AAT AAG ATG CAC TCG CCG AAG AAC ACG CTG CGT AAG 1824 Pro Pro Pro Pro Asn Lys Met His Ser Pro Lys Asn Thr Leu Arg Lys 595 600 605 CCC CCG ATG CAC CAG ATG CAC CAG GGT CCC AAC TCG GAG ACC CTC TTC 1872 Pro Pro Met His Gln Met His Gln Gly Pro Asn Ser Glu Thr Leu Phe 610 615 620 CAG TTC CAC GTG ACG GCT ACA ACA CCC AGC AGT CGT ATC GTG GTC GCG 1920 Gln Phe His Val Thr Ala Thr Thr Pro Ser Ser Arg Ile Val Val Ala 625 630 635 640 ACA ACT TCG GAA CAC TGC GTT CCC ACC AGG TGATGGGCGA CAATTACAGG 1970 Thr Thr Ser Glu His Cys Val Pro Thr Arg 645 650 CGCGGCGATG GCTTTTCCAC CACCCGCAGC GTCAAGAAGG TTTACCTTTG AGACGGGAGT 2030 GGGGCGGCTG AAACCAGTCA GGGACTAATT ACCCAAAATA TGGCTGTAAA CAACACAAAC 2090 ACACGTAACA GAAGTCTTGG TCGCGCAAGA AGACAGCCGC CCCGTCATGG CATTGTAACT 2150 CAACACCGCT CGAATAGCCC CCAGCAGCAG CAGCAGCAGT CGCAGCAGCC GCACTCCAGT 2210 TCGGGCTCCT CGCCCGTAAT GTCCAACAGC AGCAGCAGTC CGGCTCCGCC CTCCAGCAGT 2270 CCCAGTCCGC AGGAGAGCCC CAAGAACTGC AGCTACATCT ACCGTGATTG ATTGATATGC 2330 AACACCAAAT CGATGCCACT CATCCAGGCC CAGTCCACGC ACGCCCAGCC ACACTCACAC 2390 CCGCACCCGC ACCCGCTTCC GCCACCCGGT CCGACCACGC CCCCAGCACA GCCACGCGCC 2450 AGAAGTCCAA TGATCGGCAG GACATATGCC AAGTCCATGC CCGTGACACC AGTTCAACCG 2510 CAATCGCCGC TGGCTGAGAC GCCCTCCTAT GAGCTCTACG AACGCCACTC GGATGCGGCC 2570 ACCTTCCACT TTGGGGATGA GGACGATGAC GATGATGATG AGCACGACCA GGAGGACACC 2630 TCATCGCTGG CCATGATCAC ACCGCCGCCG CCCTACGACA CTCCGCATCT GATTGCATCG 2690 CCACCGCTGC CGCCGCCTCG TAGATTTCGC TTTGGCAACA GGGAGCTGTT CAGCATGAGT 2750 CCAGCCGGAG GTGGAACCAC GCCCACCGCC TCGGCAGGCC AACGCGGCAG CAGCGCCATC 2810 ACGCCCACAA AGTTGAGTGC GGCGGCAGCG GCCATGTTTG CCGCACCCCA AATGGCCACC 2870 CAACTCAACC GGAAGTGGGC TCATTTGCAA AGGAAGCGGC GCAGGCGCAA CAGCAGCTCC 2930 GGCGATTCTA AGGAGCTCGA CAAACTGGTC CTGCAATCGG TCGACTGGGA TGAGAATGAG 2990 ATGTACTAGA ACGCAAACCA ACAATGAGAT AGCAGAAACA CTTTGATTCG GAATTTATAC 3050 ACCTTTGCAT ATTTTGAATA TGACTTCAAT TTTAAAATGC GTAATTATGT TCTTATTTTT 3110 TAAAGAACGC TTTAGAGAAG TTTTCTGCTA CCTTAAATAG TACACACAAC TCATATCTAA 3170 CGTGGCGCTG CGATATAGGA ATAACCACTC CCCCTTCCCT TAAACTTAAA GTAGCAATCG 3230 AAAAGATCAT TCATTAGCGA CAGAAACTGG ATGGGGATTT ACTTACACAC AAAAAGCCAG 3290 AGAAGTTATA CACGAAGTTT ATAGTTATAT AGCCTTTATA CATACTCCCC GATCTGCTAA 3350 GTATACACAA GCAAGCATAA CATAACATAC GTATATATGA CTCTATATAT ACCAATAGAT 3410 TTCATAGACG ATTCACATGG ATCGGCTACG CTAAATTAGA GCTGCAAAAT GATATTGTTA 3470 ATTACGATTA GAGAAAAAAA AAAAGGAATT CGATATCAAG CKTATCGATA CCNTCGACCT 3530 CGNNNNNGGG GCCCGGTACC CAATTCGCCC 3560 650 amino acids amino acid linear protein 60 Glu Asp Asp Cys Gln Asn Tyr Ile Arg Ile Met Val Val Pro Ser Pro 1 5 10 15 Gly Arg Leu Phe Val Cys Gly Thr Asn Ser Phe Arg Pro Met Cys Asn 20 25 30 Thr Tyr Ile Ile Ser Asp Ser Asn Tyr Thr Leu Glu Ala Thr Lys Asn 35 40 45 Gly Gln Ala Val Cys Pro Tyr Asp Pro Arg His Asn Ser Thr Ser Val 50 55 60 Leu Ala Asp Asn Glu Leu Tyr Ser Gly Thr Val Ala Asp Phe Ser Gly 65 70 75 80 Ser Asp Pro Ile Ile Tyr Arg Glu Pro Leu Gln Thr Glu Gln Tyr Asp 85 90 95 Ser Leu Ser Leu Asn Ala Pro Asn Phe Val Ser Ser Phe Thr Gln Gly 100 105 110 Asp Phe Val Tyr Phe Phe Phe Arg Glu Thr Ala Val Glu Phe Ile Asn 115 120 125 Cys Gly Lys Ala Ile Tyr Ser Arg Val Ala Arg Val Cys Lys Trp Asp 130 135 140 Lys Gly Gly Pro His Arg Phe Arg Asn Arg Trp Thr Ser Phe Leu Lys 145 150 155 160 Ser Arg Leu Asn Cys Ser Ile Pro Gly Asp Tyr Pro Phe Tyr Phe Asn 165 170 175 Glu Ile Gln Ser Ala Ser Asn Leu Val Glu Gly Gln Tyr Gly Ser Met 180 185 190 Ser Ser Lys Leu Ile Tyr Gly Val Phe Asn Thr Pro Ser Asn Ser Ile 195 200 205 Pro Gly Ser Ala Val Cys Ala Phe Ala Leu Gln Asp Ile Ala Asp Thr 210 215 220 Phe Glu Gly Gln Phe Lys Glu Gln Thr Gly Ile Asn Ser Asn Trp Leu 225 230 235 240 Pro Val Asn Asn Ala Lys Val Pro Asp Pro Arg Pro Gly Ser Cys His 245 250 255 Asn Asp Ser Arg Ala Leu Pro Asp Pro Thr Leu Asn Phe Ile Lys Thr 260 265 270 His Ser Leu Met Asp Glu Asn Val Pro Ala Phe Phe Ser Gln Pro Ile 275 280 285 Leu Val Arg Thr Ser Thr Ile Tyr Arg Phe Thr Gln Ile Ala Val Asp 290 295 300 Ala Gln Ile Lys Thr Pro Gly Gly Lys Thr Tyr Asp Val Ile Phe Val 305 310 315 320 Gly Thr Asp His Gly Lys Ile Ile Lys Ser Val Asn Ala Glu Ser Ala 325 330 335 Asp Ser Ala Asp Lys Val Thr Ser Val Val Ile Glu Glu Ile Asp Val 340 345 350 Leu Thr Lys Ser Glu Pro Ile Arg Asn Leu Glu Ile Val Arg Thr Met 355 360 365 Gln Tyr Asp Gln Pro Lys Asp Gly Ser Tyr Asp Asp Gly Lys Leu Ile 370 375 380 Ile Val Thr Asp Ser Gln Val Val Ala Ile Gln Leu His Arg Cys His 385 390 395 400 Asn Asp Lys Ile Thr Ser Cys Ser Glu Cys Val Ala Leu Gln Asp Pro 405 410 415 Tyr Cys Ala Trp Asp Lys Ile Ala Gly Lys Cys Arg Ser His Gly Ala 420 425 430 Pro Arg Trp Leu Glu Glu Asn Tyr Phe Tyr Gln Asn Val Ala Thr Gly 435 440 445 Gln His Ala Ala Cys Pro Ser Gly Lys Ile Asn Ser Lys Asp Ala Asn 450 455 460 Ala Gly Glu Gln Lys Gly Phe Arg Asn Asp Met Asp Leu Leu Asp Ser 465 470 475 480 Arg Arg Gln Ser Lys Asp Gln Glu Ile Ile Asp Asn Ile Asp Lys Asn 485 490 495 Phe Glu Asp Ile Ile Asn Ala Gln Tyr Thr Val Glu Thr Leu Val Met 500 505 510 Ala Val Leu Ala Gly Ser Ile Phe Ser Leu Leu Val Gly Phe Phe Thr 515 520 525 Gly Tyr Phe Cys Gly Arg Arg Cys His Lys Asp Glu Asp Asp Asn Leu 530 535 540 Pro Tyr Pro Asp Thr Glu Tyr Glu Tyr Phe Glu Gln Arg Gln Asn Val 545 550 555 560 Asn Ser Phe Pro Ser Ser Cys Arg Ile Gln Gln Glu Pro Lys Leu Leu 565 570 575 Pro Gln Val Glu Glu Val Thr Tyr Ala Asp Ala Val Leu Leu Pro Gln 580 585 590 Pro Pro Pro Pro Asn Lys Met His Ser Pro Lys Asn Thr Leu Arg Lys 595 600 605 Pro Pro Met His Gln Met His Gln Gly Pro Asn Ser Glu Thr Leu Phe 610 615 620 Gln Phe His Val Thr Ala Thr Thr Pro Ser Ser Arg Ile Val Val Ala 625 630 635 640 Thr Thr Ser Glu His Cys Val Pro Thr Arg 645 650 2670 base pairs nucleic acid double linear cDNA CDS 268..2439 61 GAAAATCGAA CWCCGAATTG AATGAACWGC AAAACGCCAA TTAGATAGTT GCAAGCCTAA 60 TGCATTTCAG AKATTTNMMC GATGCGAAAC AAGTTCCGCC ACGAAAGTGA ACAGTGGTAA 120 AATGCCCAAG AATCTCGAGC GGAAACACCA AACACAAAAG AACAAGCAAC CGCCTCTCAC 180 TCGCTCTTGC ACTTTAATCC AATTGAGGTT GGTGGGGTCG CATTCGCCCC CCGGTCGACC 240 ACCCCTCTCG CTCGCACCGC CCTCGCA ATG TCT CTT CTA CAG CTA TCG CCG CTC 294 Met Ser Leu Leu Gln Leu Ser Pro Leu 1 5 CTC GCA CTC CTG CTA CTC CTC TGC AGT AGT GTG AGC GAG ACG GCT GCG 342 Leu Ala Leu Leu Leu Leu Leu Cys Ser Ser Val Ser Glu Thr Ala Ala 10 15 20 25 GAC TAC GAG AAC ACC TGG AAC TTC TAC TAC GAG CGT CCC TGT TGC ACT 390 Asp Tyr Glu Asn Thr Trp Asn Phe Tyr Tyr Glu Arg Pro Cys Cys Thr 30 35 40 GGA AAC GAT CAG GGG AAC AAC AAT TAC GGA AAA CAC GGC GCA GAT CAT 438 Gly Asn Asp Gln Gly Asn Asn Asn Tyr Gly Lys His Gly Ala Asp His 45 50 55 GTG CGG GAG TTC AAC TGC GGC AAG CTG TAC TAT CGT ACA TTC CAT ATG 486 Val Arg Glu Phe Asn Cys Gly Lys Leu Tyr Tyr Arg Thr Phe His Met 60 65 70 AAC GAA GAT CGA GAT ACG CTC TAT GTG GGA GCC ATG GAT CGC GTA TTC 534 Asn Glu Asp Arg Asp Thr Leu Tyr Val Gly Ala Met Asp Arg Val Phe 75 80 85 CGT GTG AAC CTG CAG AAT ATC TCC TCA TCC AAT TGT AAT CGG GAT GCG 582 Arg Val Asn Leu Gln Asn Ile Ser Ser Ser Asn Cys Asn Arg Asp Ala 90 95 100 105 ATC AAC TTG GAG CCA ACA CGG GAT GAT GTG GTT AGC TGC GTC TCC AAA 630 Ile Asn Leu Glu Pro Thr Arg Asp Asp Val Val Ser Cys Val Ser Lys 110 115 120 GGC AAA AGT CAG ATC TTC GAC TGC AAG AAC CAT GTG CGT GTC ATC CAG 678 Gly Lys Ser Gln Ile Phe Asp Cys Lys Asn His Val Arg Val Ile Gln 125 130 135 TCA ATG GAC CAG GGG GAT AGG CTC TAT GTA TGC GGC ACC AAC GCC CAC 726 Ser Met Asp Gln Gly Asp Arg Leu Tyr Val Cys Gly Thr Asn Ala His 140 145 150 AAT CCC AAG GAT TAT GTT ATC TAT GCG AAT CTA ACC CAC CTG CCG CGC 774 Asn Pro Lys Asp Tyr Val Ile Tyr Ala Asn Leu Thr His Leu Pro Arg 155 160 165 TCG GAA TAT GTG ATT GGC GTG GGT CTG GGC ATT GCC AAG TGC CCC TAC 822 Ser Glu Tyr Val Ile Gly Val Gly Leu Gly Ile Ala Lys Cys Pro Tyr 170 175 180 185 GAT CCC CTC GAC AAC TCA ACT GCG ATT TAT GTG GAG AAT GGC AAT CCG 870 Asp Pro Leu Asp Asn Ser Thr Ala Ile Tyr Val Glu Asn Gly Asn Pro 190 195 200 GGT GGT CTG CCC GGT TTG TAC TCC GGC ACC AAT GCG GAG TTC ACC AAG 918 Gly Gly Leu Pro Gly Leu Tyr Ser Gly Thr Asn Ala Glu Phe Thr Lys 205 210 215 GCG GAT ACG GTT ATT TTC CGC ACT GAT CTG TAT AAT ACT TCG GCT AAA 966 Ala Asp Thr Val Ile Phe Arg Thr Asp Leu Tyr Asn Thr Ser Ala Lys 220 225 230 CGT TTG GAA TAT AAA TTC AAG AGG ACT CTG AAA TAC GAC TCC AAG TGG 1014 Arg Leu Glu Tyr Lys Phe Lys Arg Thr Leu Lys Tyr Asp Ser Lys Trp 235 240 245 TTG GAC AAA CCA AAC TTT GTC GGC TCC TTT GAT ATT GGG GAG TAC GTG 1062 Leu Asp Lys Pro Asn Phe Val Gly Ser Phe Asp Ile Gly Glu Tyr Val 250 255 260 265 TAT TTC TTT TTC CGT GAA ACC GCC GTG GAA TAC ATC AAC TGC GGC AAG 1110 Tyr Phe Phe Phe Arg Glu Thr Ala Val Glu Tyr Ile Asn Cys Gly Lys 270 275 280 GCT GTC TAT TCG CGC ATC GCA CGG GTG TGC AAG AAG GAT GTG GGT GGA 1158 Ala Val Tyr Ser Arg Ile Ala Arg Val Cys Lys Lys Asp Val Gly Gly 285 290 295 AAG AAT CTG CTG GCC CAC AAC TGG GCC ACC TAC CTG AAG GCC AGA CTC 1206 Lys Asn Leu Leu Ala His Asn Trp Ala Thr Tyr Leu Lys Ala Arg Leu 300 305 310 AAC TGC AGC ATC TCC GGC GAA TTT CCG TTC TAT TTC AAC GAG ATC CAA 1254 Asn Cys Ser Ile Ser Gly Glu Phe Pro Phe Tyr Phe Asn Glu Ile Gln 315 320 325 TCG GTC TAC CAG CTG CCC TCC GAT AAG AGT CGA TTC TTC GCC ACA TTC 1302 Ser Val Tyr Gln Leu Pro Ser Asp Lys Ser Arg Phe Phe Ala Thr Phe 330 335 340 345 ACG ACG AGC ACT AAT GGC CTG ATT GGA TCT GCC GTA TGC AGT TTC CAC 1350 Thr Thr Ser Thr Asn Gly Leu Ile Gly Ser Ala Val Cys Ser Phe His 350 355 360 ATT AAC GAG ATT CAG GCT GCC TTC AAT GGC AAA TTC AAG GAG CAA TCT 1398 Ile Asn Glu Ile Gln Ala Ala Phe Asn Gly Lys Phe Lys Glu Gln Ser 365 370 375 TCA TCG AAT TCC GCA TGG CTG CCG GTG CTT AAC TCC CGG GTG CCG GAA 1446 Ser Ser Asn Ser Ala Trp Leu Pro Val Leu Asn Ser Arg Val Pro Glu 380 385 390 CCA CGG CCG GGT ACA TGT GTC AAC GAT ACA TCA AAC CTG CCC GAT ACC 1494 Pro Arg Pro Gly Thr Cys Val Asn Asp Thr Ser Asn Leu Pro Asp Thr 395 400 405 GTA CTG AAT TTC ATC AGA TCC CAT CCA CTT ATG GAC AAA GCC GTA AAT 1542 Val Leu Asn Phe Ile Arg Ser His Pro Leu Met Asp Lys Ala Val Asn 410 415 420 425 CAC GAG CAC AAC AAT CCA GTC TAT TAT AAA AGG GAT TTG GTC TTC ACC 1590 His Glu His Asn Asn Pro Val Tyr Tyr Lys Arg Asp Leu Val Phe Thr 430 435 440 AAG CTC GTC GTT GAC AAA ATT CGC ATT GAC ATC CTC AAC CAG GAA TAC 1638 Lys Leu Val Val Asp Lys Ile Arg Ile Asp Ile Leu Asn Gln Glu Tyr 445 450 455 ATT GTG TAC TAT GTG GGC ACC AAT CTG GGT CGC ATT TAC AAA ATC GTG 1686 Ile Val Tyr Tyr Val Gly Thr Asn Leu Gly Arg Ile Tyr Lys Ile Val 460 465 470 CAG TAC TAC CGT AAC GGA GAG TCG CTG TCC AAG CTT CTG GAT ATC TTC 1734 Gln Tyr Tyr Arg Asn Gly Glu Ser Leu Ser Lys Leu Leu Asp Ile Phe 475 480 485 GAG GTG GCT CCA AAC GAG GCC ATC CAA GTG ATG GAA ATC AGC CAG ACA 1782 Glu Val Ala Pro Asn Glu Ala Ile Gln Val Met Glu Ile Ser Gln Thr 490 495 500 505 CGT AAG AGC CTC TAC ATT GGC ACC GAT CAT CGC ATC AAG CAA ATC GAC 1830 Arg Lys Ser Leu Tyr Ile Gly Thr Asp His Arg Ile Lys Gln Ile Asp 510 515 520 CTG GCC ATG TGC AAT CGC CGT TAC GAC AAC TGC TTC CGC TGC GTC CGT 1878 Leu Ala Met Cys Asn Arg Arg Tyr Asp Asn Cys Phe Arg Cys Val Arg 525 530 535 GAT CCC TAC TGC GGC TGG GAT AAG GAG GCC AAT ACG TGC CGA CCG TAC 1926 Asp Pro Tyr Cys Gly Trp Asp Lys Glu Ala Asn Thr Cys Arg Pro Tyr 540 545 550 GAG CTG GAT TTA CTG CAG GAT GTG GCC AAT GAA ACG AGT GAC ATT TGC 1974 Glu Leu Asp Leu Leu Gln Asp Val Ala Asn Glu Thr Ser Asp Ile Cys 555 560 565 GAT TCG AGT GTG CTG AAA AAG AAG ATT GTG GTG ACC TAT GGC CAG AGT 2022 Asp Ser Ser Val Leu Lys Lys Lys Ile Val Val Thr Tyr Gly Gln Ser 570 575 580 585 GTA CAT CTG GGC TGT TTC GTC AAA ATA CCC GAA GTG CTG AAG AAT GAG 2070 Val His Leu Gly Cys Phe Val Lys Ile Pro Glu Val Leu Lys Asn Glu 590 595 600 CAA GTG ACC TGG TAT CAT CAC TCC AAG GAC AAG GGA CGC TAC GAG ATT 2118 Gln Val Thr Trp Tyr His His Ser Lys Asp Lys Gly Arg Tyr Glu Ile 605 610 615 CGT TAC TCG CCG ACC AAA TAC ATT GAG ACC ACC GAA CGT GGC CTG GTT 2166 Arg Tyr Ser Pro Thr Lys Tyr Ile Glu Thr Thr Glu Arg Gly Leu Val 620 625 630 GTG GTT TCC GTG AAC GAA GCC GAT GGT GGT CGG TAC GAT TGC CAT TTG 2214 Val Val Ser Val Asn Glu Ala Asp Gly Gly Arg Tyr Asp Cys His Leu 635 640 645 GGC GGC TCG CTT TTG TGC AGC TAC AAC ATT ACA GTG GAT GCC CAC AGA 2262 Gly Gly Ser Leu Leu Cys Ser Tyr Asn Ile Thr Val Asp Ala His Arg 650 655 660 665 TGC ACT CCG CCG AAC AAG AGT AAT GAC TAT CAG AAA ATC TAC TCG GAC 2310 Cys Thr Pro Pro Asn Lys Ser Asn Asp Tyr Gln Lys Ile Tyr Ser Asp 670 675 680 TGG TGC CAC GAG TTC GAG AAA TAC AAA ACA GCA ATG AAG TCC TGG GAA 2358 Trp Cys His Glu Phe Glu Lys Tyr Lys Thr Ala Met Lys Ser Trp Glu 685 690 695 AAG AAG CAA GGC CAA TGC TCG ACA CGG CAG AAC TTC AGC TGC AAT CAG 2406 Lys Lys Gln Gly Gln Cys Ser Thr Arg Gln Asn Phe Ser Cys Asn Gln 700 705 710 CAT CCG AAT GAG ATT TTC CGT AAG CCC AAT GTC TGATATCACG AAGAGAGTAT 2459 His Pro Asn Glu Ile Phe Arg Lys Pro Asn Val 715 720 CGCCCTCAAA ATGCCGTCAT CGTCGTCCAA TCAATTTTAG TTAATCGAAA GCGAAGAGGA 2519 TAATAACAGT GCGGAATAGA AAGCCCAGGA CGAGAAGAAC TCATTATAAT CATTATTATC 2579 AGCGACATCA TCATAGACAT ACTTTCTTCA GCAATGAACA GAAAACTCTT CCTAAAGGAT 2639 TATGCATTTA CCGAAGCATT TACAATGCAT C 2670 724 amino acids amino acid linear protein 62 Met Ser Leu Leu Gln Leu Ser Pro Leu Leu Ala Leu Leu Leu Leu Leu 1 5 10 15 Cys Ser Ser Val Ser Glu Thr Ala Ala Asp Tyr Glu Asn Thr Trp Asn 20 25 30 Phe Tyr Tyr Glu Arg Pro Cys Cys Thr Gly Asn Asp Gln Gly Asn Asn 35 40 45 Asn Tyr Gly Lys His Gly Ala Asp His Val Arg Glu Phe Asn Cys Gly 50 55 60 Lys Leu Tyr Tyr Arg Thr Phe His Met Asn Glu Asp Arg Asp Thr Leu 65 70 75 80 Tyr Val Gly Ala Met Asp Arg Val Phe Arg Val Asn Leu Gln Asn Ile 85 90 95 Ser Ser Ser Asn Cys Asn Arg Asp Ala Ile Asn Leu Glu Pro Thr Arg 100 105 110 Asp Asp Val Val Ser Cys Val Ser Lys Gly Lys Ser Gln Ile Phe Asp 115 120 125 Cys Lys Asn His Val Arg Val Ile Gln Ser Met Asp Gln Gly Asp Arg 130 135 140 Leu Tyr Val Cys Gly Thr Asn Ala His Asn Pro Lys Asp Tyr Val Ile 145 150 155 160 Tyr Ala Asn Leu Thr His Leu Pro Arg Ser Glu Tyr Val Ile Gly Val 165 170 175 Gly Leu Gly Ile Ala Lys Cys Pro Tyr Asp Pro Leu Asp Asn Ser Thr 180 185 190 Ala Ile Tyr Val Glu Asn Gly Asn Pro Gly Gly Leu Pro Gly Leu Tyr 195 200 205 Ser Gly Thr Asn Ala Glu Phe Thr Lys Ala Asp Thr Val Ile Phe Arg 210 215 220 Thr Asp Leu Tyr Asn Thr Ser Ala Lys Arg Leu Glu Tyr Lys Phe Lys 225 230 235 240 Arg Thr Leu Lys Tyr Asp Ser Lys Trp Leu Asp Lys Pro Asn Phe Val 245 250 255 Gly Ser Phe Asp Ile Gly Glu Tyr Val Tyr Phe Phe Phe Arg Glu Thr 260 265 270 Ala Val Glu Tyr Ile Asn Cys Gly Lys Ala Val Tyr Ser Arg Ile Ala 275 280 285 Arg Val Cys Lys Lys Asp Val Gly Gly Lys Asn Leu Leu Ala His Asn 290 295 300 Trp Ala Thr Tyr Leu Lys Ala Arg Leu Asn Cys Ser Ile Ser Gly Glu 305 310 315 320 Phe Pro Phe Tyr Phe Asn Glu Ile Gln Ser Val Tyr Gln Leu Pro Ser 325 330 335 Asp Lys Ser Arg Phe Phe Ala Thr Phe Thr Thr Ser Thr Asn Gly Leu 340 345 350 Ile Gly Ser Ala Val Cys Ser Phe His Ile Asn Glu Ile Gln Ala Ala 355 360 365 Phe Asn Gly Lys Phe Lys Glu Gln Ser Ser Ser Asn Ser Ala Trp Leu 370 375 380 Pro Val Leu Asn Ser Arg Val Pro Glu Pro Arg Pro Gly Thr Cys Val 385 390 395 400 Asn Asp Thr Ser Asn Leu Pro Asp Thr Val Leu Asn Phe Ile Arg Ser 405 410 415 His Pro Leu Met Asp Lys Ala Val Asn His Glu His Asn Asn Pro Val 420 425 430 Tyr Tyr Lys Arg Asp Leu Val Phe Thr Lys Leu Val Val Asp Lys Ile 435 440 445 Arg Ile Asp Ile Leu Asn Gln Glu Tyr Ile Val Tyr Tyr Val Gly Thr 450 455 460 Asn Leu Gly Arg Ile Tyr Lys Ile Val Gln Tyr Tyr Arg Asn Gly Glu 465 470 475 480 Ser Leu Ser Lys Leu Leu Asp Ile Phe Glu Val Ala Pro Asn Glu Ala 485 490 495 Ile Gln Val Met Glu Ile Ser Gln Thr Arg Lys Ser Leu Tyr Ile Gly 500 505 510 Thr Asp His Arg Ile Lys Gln Ile Asp Leu Ala Met Cys Asn Arg Arg 515 520 525 Tyr Asp Asn Cys Phe Arg Cys Val Arg Asp Pro Tyr Cys Gly Trp Asp 530 535 540 Lys Glu Ala Asn Thr Cys Arg Pro Tyr Glu Leu Asp Leu Leu Gln Asp 545 550 555 560 Val Ala Asn Glu Thr Ser Asp Ile Cys Asp Ser Ser Val Leu Lys Lys 565 570 575 Lys Ile Val Val Thr Tyr Gly Gln Ser Val His Leu Gly Cys Phe Val 580 585 590 Lys Ile Pro Glu Val Leu Lys Asn Glu Gln Val Thr Trp Tyr His His 595 600 605 Ser Lys Asp Lys Gly Arg Tyr Glu Ile Arg Tyr Ser Pro Thr Lys Tyr 610 615 620 Ile Glu Thr Thr Glu Arg Gly Leu Val Val Val Ser Val Asn Glu Ala 625 630 635 640 Asp Gly Gly Arg Tyr Asp Cys His Leu Gly Gly Ser Leu Leu Cys Ser 645 650 655 Tyr Asn Ile Thr Val Asp Ala His Arg Cys Thr Pro Pro Asn Lys Ser 660 665 670 Asn Asp Tyr Gln Lys Ile Tyr Ser Asp Trp Cys His Glu Phe Glu Lys 675 680 685 Tyr Lys Thr Ala Met Lys Ser Trp Glu Lys Lys Gln Gly Gln Cys Ser 690 695 700 Thr Arg Gln Asn Phe Ser Cys Asn Gln His Pro Asn Glu Ile Phe Arg 705 710 715 720 Lys Pro Asn Val 2504 base pairs nucleic acid double linear cDNA CDS 355..2493 63 GGCCGGTCGA CCACGAGCGA AGTTTAGTAT CAAGTTGAGA GTTTGTTTGG AGCGTAGTTT 60 ACGGAGCGTA CATTTAAATT TGCGGACAAA TCGTGTTTTG GTGCTTCTCT GTGGATTGTT 120 GTGTTCTTGA AGATGCTTCC CTTGGTTTTC GGATAAGCTT TCCTGTGGAT TGTTGTGTTC 180 TTGAAGATGC TTCCCTTGGT TTTCGGATAA GCTTTCCAGC GTGGTTTCAG CCTCGGCTTG 240 TTTGGACCCC GACATAATCT TCGAACTACA ATGAAGAGGA AATTTTGAAA CGCGTTTCAG 300 ACGCGTACAA TCGACAAAAT GTTTGGTTTC CAATTGATCT TGCAATGTAG CTAC ATG 357 Met 1 GTG GTG AAG ATC TTG GTT TGG TCG ATA TGT CTG ATA GCG CTG TGT CAT 405 Val Val Lys Ile Leu Val Trp Ser Ile Cys Leu Ile Ala Leu Cys His 5 10 15 GCT TGG ATG CCG GAT AGT TCT TCC AAA TTA ATA AAC CAT TTT AAA TCA 453 Ala Trp Met Pro Asp Ser Ser Ser Lys Leu Ile Asn His Phe Lys Ser 20 25 30 GTT GAA AGT AAA AGC TTT ACC GGG AAC GCC ACG TTC CCT GAT CAC TTT 501 Val Glu Ser Lys Ser Phe Thr Gly Asn Ala Thr Phe Pro Asp His Phe 35 40 45 ATT GTC TTG AAT CAA GAC GAA ACT TCG ATA TTA GTA GGC GGT AGA AAT 549 Ile Val Leu Asn Gln Asp Glu Thr Ser Ile Leu Val Gly Gly Arg Asn 50 55 60 65 AGG GTT TAC AAT TTA AGT ATA TTC GAC CTC AGT GAG CGT AAA GGG GGG 597 Arg Val Tyr Asn Leu Ser Ile Phe Asp Leu Ser Glu Arg Lys Gly Gly 70 75 80 CGA ATC GAC TGG CCA TCG TCC GAT GCA CAT GGC CAG TTG TGT ATA TTG 645 Arg Ile Asp Trp Pro Ser Ser Asp Ala His Gly Gln Leu Cys Ile Leu 85 90 95 AAA GGG AAA ACG GAC GAC GAC TGC CAA AAT TAC ATT AGA ATA CTG TAC 693 Lys Gly Lys Thr Asp Asp Asp Cys Gln Asn Tyr Ile Arg Ile Leu Tyr 100 105 110 TCT TCA GAA CCG GGG AAA TTA GTT ATT TGC GGG ACC AAT TCG TAC AAA 741 Ser Ser Glu Pro Gly Lys Leu Val Ile Cys Gly Thr Asn Ser Tyr Lys 115 120 125 CCC CTC TGT CGG ACG TAC GCA TTT AAG GAG GGA AAG TAC CTG GTT GAG 789 Pro Leu Cys Arg Thr Tyr Ala Phe Lys Glu Gly Lys Tyr Leu Val Glu 130 135 140 145 AAA GAA GTA GAA GGG ATA GGC TTG TGT CCA TAC AAT CCG GAA CAC AAC 837 Lys Glu Val Glu Gly Ile Gly Leu Cys Pro Tyr Asn Pro Glu His Asn 150 155 160 AGC ACA TCT GTC TCC TAC AAT GGC CAA TTA TTT TCA GCG ACG GTC GCC 885 Ser Thr Ser Val Ser Tyr Asn Gly Gln Leu Phe Ser Ala Thr Val Ala 165 170 175 GAC TTT TCC GGG GGC GAC CCT CTC ATA TAC AGG GAG CCC CAG CGC ACC 933 Asp Phe Ser Gly Gly Asp Pro Leu Ile Tyr Arg Glu Pro Gln Arg Thr 180 185 190 GAA CTC TCA GAT CTC AAA CAA CTG AAC GCA CCG AAT TTC GTA AAC TCG 981 Glu Leu Ser Asp Leu Lys Gln Leu Asn Ala Pro Asn Phe Val Asn Ser 195 200 205 GTG GCC TAT GGC GAC TAC ATA TTC TTC TTC TAC CGT GAA ACC GCC GTC 1029 Val Ala Tyr Gly Asp Tyr Ile Phe Phe Phe Tyr Arg Glu Thr Ala Val 210 215 220 225 GAG TAC ATG AAC TGC GGA AAA GTC ATC TAC TCG CGG GTC GCC AGG GTG 1077 Glu Tyr Met Asn Cys Gly Lys Val Ile Tyr Ser Arg Val Ala Arg Val 230 235 240 TGC AAG GAC GAC AAA GGG GGC CCT CAC CAG TCA CGC GAC CGC TGG ACG 1125 Cys Lys Asp Asp Lys Gly Gly Pro His Gln Ser Arg Asp Arg Trp Thr 245 250 255 TCG TTC CTC AAA GCA CGT CTC AAT TGT TCA ATT CCC GGC GAG TAC CCC 1173 Ser Phe Leu Lys Ala Arg Leu Asn Cys Ser Ile Pro Gly Glu Tyr Pro 260 265 270 TTT TAC TTT GAT GAA ATC CAA TCA ACA AGT GAT ATA GTC GAG GGT CGG 1221 Phe Tyr Phe Asp Glu Ile Gln Ser Thr Ser Asp Ile Val Glu Gly Arg 275 280 285 TAC AAT TCC GAC GAC AGC AAA AAG ATC ATT TAT GGA ATC CTC ACA ACT 1269 Tyr Asn Ser Asp Asp Ser Lys Lys Ile Ile Tyr Gly Ile Leu Thr Thr 290 295 300 305 CCA GTT AAT GCC ATC GGC GGC TCG GCC ATT TGC GCG TAT CAA ATG GCC 1317 Pro Val Asn Ala Ile Gly Gly Ser Ala Ile Cys Ala Tyr Gln Met Ala 310 315 320 GAC ATC TTG CGC GTG TTT GAA GGG AGC TTC AAG CAC CAA GAG ACG ATC 1365 Asp Ile Leu Arg Val Phe Glu Gly Ser Phe Lys His Gln Glu Thr Ile 325 330 335 AAC TCG AAC TGG CTC CCC GTG CCC CAG AAC CTA GTC CCT GAA CCC AGG 1413 Asn Ser Asn Trp Leu Pro Val Pro Gln Asn Leu Val Pro Glu Pro Arg 340 345 350 CCC GGG CAG TGC GTA CGC GAC AGC AGG ATC CTG CCC GAC AAG AAC GTC 1461 Pro Gly Gln Cys Val Arg Asp Ser Arg Ile Leu Pro Asp Lys Asn Val 355 360 365 AAC TTT ATT AAG ACC CAC TCT TTG ATG GAG GAC GTT CCG GCT CTT TTC 1509 Asn Phe Ile Lys Thr His Ser Leu Met Glu Asp Val Pro Ala Leu Phe 370 375 380 385 GGA AAA CCA GTT CTG GTC CGA GTG AGT CTG CAG TAT CGG TTT ACA GCC 1557 Gly Lys Pro Val Leu Val Arg Val Ser Leu Gln Tyr Arg Phe Thr Ala 390 395 400 ATA ACA GTG GAT CCA CAA GTG AAA ACA ATC AAT AAT CAG TAT CTC GAT 1605 Ile Thr Val Asp Pro Gln Val Lys Thr Ile Asn Asn Gln Tyr Leu Asp 405 410 415 GTT TTG TAT ATC GGA ACA GAT GAT GGG AAG GTA CTA AAA GCT GTT AAT 1653 Val Leu Tyr Ile Gly Thr Asp Asp Gly Lys Val Leu Lys Ala Val Asn 420 425 430 ATA CCA AAG CGA CAC GCT AAA GCG TTG TTA TAT CGA AAA TAC CGT ACA 1701 Ile Pro Lys Arg His Ala Lys Ala Leu Leu Tyr Arg Lys Tyr Arg Thr 435 440 445 TCC GTA CAT CCG CAC GGA GCT CCC GTA AAA CAG CTG AAG ATC GCT CCC 1749 Ser Val His Pro His Gly Ala Pro Val Lys Gln Leu Lys Ile Ala Pro 450 455 460 465 GGT TAT GGC AAA GTT GTG GTG GTC GGG AAA GAC GAA ATC AGA CTT GCT 1797 Gly Tyr Gly Lys Val Val Val Val Gly Lys Asp Glu Ile Arg Leu Ala 470 475 480 AAT CTC AAC CAT TGT GCA AGC AAA ACG CGG TGC AAG GAC TGT GTG GAA 1845 Asn Leu Asn His Cys Ala Ser Lys Thr Arg Cys Lys Asp Cys Val Glu 485 490 495 CTG CAA GAC CCA CAT TGC GCC TGG GAC GCC AAA CAA AAC CTG TGT GTC 1893 Leu Gln Asp Pro His Cys Ala Trp Asp Ala Lys Gln Asn Leu Cys Val 500 505 510 AGC ATT GAC ACC GTC ACT TCG TAT CGC TTC CTG ATC CAG GAC GTA GTT 1941 Ser Ile Asp Thr Val Thr Ser Tyr Arg Phe Leu Ile Gln Asp Val Val 515 520 525 CGC GGC GAC GAC AAC AAA TGT TGG TCG CCG CAA ACA GAC AAA AAG ACT 1989 Arg Gly Asp Asp Asn Lys Cys Trp Ser Pro Gln Thr Asp Lys Lys Thr 530 535 540 545 GTG ATT AAG AAT AAG CCC AGC GAG GTT GAG AAC GAG ATT ACG AAC TCC 2037 Val Ile Lys Asn Lys Pro Ser Glu Val Glu Asn Glu Ile Thr Asn Ser 550 555 560 ATT GAC GAA AAG GAT CTC GAT TCA AGC GAT CCG CTC ATC AAA ACT GGT 2085 Ile Asp Glu Lys Asp Leu Asp Ser Ser Asp Pro Leu Ile Lys Thr Gly 565 570 575 CTC GAT GAC GAT TCC GAT TGT GAT CCA GTC AGC GAG AAC AGC ATA GGC 2133 Leu Asp Asp Asp Ser Asp Cys Asp Pro Val Ser Glu Asn Ser Ile Gly 580 585 590 GGA TGC GCC GTC CGC CAG CAA CTT GTT ATA TAC ACA GCT GGG ACT CTA 2181 Gly Cys Ala Val Arg Gln Gln Leu Val Ile Tyr Thr Ala Gly Thr Leu 595 600 605 CAC ATT GTC GTG GTC GTC GTC AGC ATC GTG GGT TTA TTT TCT TGG CTT 2229 His Ile Val Val Val Val Val Ser Ile Val Gly Leu Phe Ser Trp Leu 610 615 620 625 TAT AGC GGG TTA TCT GTT TTC GCA AAA TTT CAC TCG GAT TCG CAA TAT 2277 Tyr Ser Gly Leu Ser Val Phe Ala Lys Phe His Ser Asp Ser Gln Tyr 630 635 640 CCT GAG GCG CCG TTT ATA GAG CAG CAC AAT CAT TTG GAA AGA TTA AGC 2325 Pro Glu Ala Pro Phe Ile Glu Gln His Asn His Leu Glu Arg Leu Ser 645 650 655 GCC AAC CAG ACG GGG TAT TTG ACT CCG AGG GCC AAT AAA GCG GTC AAT 2373 Ala Asn Gln Thr Gly Tyr Leu Thr Pro Arg Ala Asn Lys Ala Val Asn 660 665 670 TTG GTG GTG AAG GTG TCT AGT AGC ACG CCG CGG CCG AAA AAG GAC AAT 2421 Leu Val Val Lys Val Ser Ser Ser Thr Pro Arg Pro Lys Lys Asp Asn 675 680 685 CTC GAT GTC AGC AAA GAC TTG AAC ATT GCG AGT GAC GGG ACT TTG CAA 2469 Leu Asp Val Ser Lys Asp Leu Asn Ile Ala Ser Asp Gly Thr Leu Gln 690 695 700 705 AAA ATC AAG AAG ACT TAC ATT TAGTGCGACT TTTT 2504 Lys Ile Lys Lys Thr Tyr Ile 710 712 amino acids amino acid linear protein 64 Met Val Val Lys Ile Leu Val Trp Ser Ile Cys Leu Ile Ala Leu Cys 1 5 10 15 His Ala Trp Met Pro Asp Ser Ser Ser Lys Leu Ile Asn His Phe Lys 20 25 30 Ser Val Glu Ser Lys Ser Phe Thr Gly Asn Ala Thr Phe Pro Asp His 35 40 45 Phe Ile Val Leu Asn Gln Asp Glu Thr Ser Ile Leu Val Gly Gly Arg 50 55 60 Asn Arg Val Tyr Asn Leu Ser Ile Phe Asp Leu Ser Glu Arg Lys Gly 65 70 75 80 Gly Arg Ile Asp Trp Pro Ser Ser Asp Ala His Gly Gln Leu Cys Ile 85 90 95 Leu Lys Gly Lys Thr Asp Asp Asp Cys Gln Asn Tyr Ile Arg Ile Leu 100 105 110 Tyr Ser Ser Glu Pro Gly Lys Leu Val Ile Cys Gly Thr Asn Ser Tyr 115 120 125 Lys Pro Leu Cys Arg Thr Tyr Ala Phe Lys Glu Gly Lys Tyr Leu Val 130 135 140 Glu Lys Glu Val Glu Gly Ile Gly Leu Cys Pro Tyr Asn Pro Glu His 145 150 155 160 Asn Ser Thr Ser Val Ser Tyr Asn Gly Gln Leu Phe Ser Ala Thr Val 165 170 175 Ala Asp Phe Ser Gly Gly Asp Pro Leu Ile Tyr Arg Glu Pro Gln Arg 180 185 190 Thr Glu Leu Ser Asp Leu Lys Gln Leu Asn Ala Pro Asn Phe Val Asn 195 200 205 Ser Val Ala Tyr Gly Asp Tyr Ile Phe Phe Phe Tyr Arg Glu Thr Ala 210 215 220 Val Glu Tyr Met Asn Cys Gly Lys Val Ile Tyr Ser Arg Val Ala Arg 225 230 235 240 Val Cys Lys Asp Asp Lys Gly Gly Pro His Gln Ser Arg Asp Arg Trp 245 250 255 Thr Ser Phe Leu Lys Ala Arg Leu Asn Cys Ser Ile Pro Gly Glu Tyr 260 265 270 Pro Phe Tyr Phe Asp Glu Ile Gln Ser Thr Ser Asp Ile Val Glu Gly 275 280 285 Arg Tyr Asn Ser Asp Asp Ser Lys Lys Ile Ile Tyr Gly Ile Leu Thr 290 295 300 Thr Pro Val Asn Ala Ile Gly Gly Ser Ala Ile Cys Ala Tyr Gln Met 305 310 315 320 Ala Asp Ile Leu Arg Val Phe Glu Gly Ser Phe Lys His Gln Glu Thr 325 330 335 Ile Asn Ser Asn Trp Leu Pro Val Pro Gln Asn Leu Val Pro Glu Pro 340 345 350 Arg Pro Gly Gln Cys Val Arg Asp Ser Arg Ile Leu Pro Asp Lys Asn 355 360 365 Val Asn Phe Ile Lys Thr His Ser Leu Met Glu Asp Val Pro Ala Leu 370 375 380 Phe Gly Lys Pro Val Leu Val Arg Val Ser Leu Gln Tyr Arg Phe Thr 385 390 395 400 Ala Ile Thr Val Asp Pro Gln Val Lys Thr Ile Asn Asn Gln Tyr Leu 405 410 415 Asp Val Leu Tyr Ile Gly Thr Asp Asp Gly Lys Val Leu Lys Ala Val 420 425 430 Asn Ile Pro Lys Arg His Ala Lys Ala Leu Leu Tyr Arg Lys Tyr Arg 435 440 445 Thr Ser Val His Pro His Gly Ala Pro Val Lys Gln Leu Lys Ile Ala 450 455 460 Pro Gly Tyr Gly Lys Val Val Val Val Gly Lys Asp Glu Ile Arg Leu 465 470 475 480 Ala Asn Leu Asn His Cys Ala Ser Lys Thr Arg Cys Lys Asp Cys Val 485 490 495 Glu Leu Gln Asp Pro His Cys Ala Trp Asp Ala Lys Gln Asn Leu Cys 500 505 510 Val Ser Ile Asp Thr Val Thr Ser Tyr Arg Phe Leu Ile Gln Asp Val 515 520 525 Val Arg Gly Asp Asp Asn Lys Cys Trp Ser Pro Gln Thr Asp Lys Lys 530 535 540 Thr Val Ile Lys Asn Lys Pro Ser Glu Val Glu Asn Glu Ile Thr Asn 545 550 555 560 Ser Ile Asp Glu Lys Asp Leu Asp Ser Ser Asp Pro Leu Ile Lys Thr 565 570 575 Gly Leu Asp Asp Asp Ser Asp Cys Asp Pro Val Ser Glu Asn Ser Ile 580 585 590 Gly Gly Cys Ala Val Arg Gln Gln Leu Val Ile Tyr Thr Ala Gly Thr 595 600 605 Leu His Ile Val Val Val Val Val Ser Ile Val Gly Leu Phe Ser Trp 610 615 620 Leu Tyr Ser Gly Leu Ser Val Phe Ala Lys Phe His Ser Asp Ser Gln 625 630 635 640 Tyr Pro Glu Ala Pro Phe Ile Glu Gln His Asn His Leu Glu Arg Leu 645 650 655 Ser Ala Asn Gln Thr Gly Tyr Leu Thr Pro Arg Ala Asn Lys Ala Val 660 665 670 Asn Leu Val Val Lys Val Ser Ser Ser Thr Pro Arg Pro Lys Lys Asp 675 680 685 Asn Leu Asp Val Ser Lys Asp Leu Asn Ile Ala Ser Asp Gly Thr Leu 690 695 700 Gln Lys Ile Lys Lys Thr Tyr Ile 705 710 369 base pairs nucleic acid double linear cDNA CDS 1..369 65 ATG ATT TAT TTA TAC ACG GCG GAT AAC GTA ATT CCA AAA GAT GGT TTA 48 Met Ile Tyr Leu Tyr Thr Ala Asp Asn Val Ile Pro Lys Asp Gly Leu 1 5 10 15 CAA GGA GCA TTT GTC GAT AAA GAC GGT ACT TAT GAC AAA GTT TAC ATT 96 Gln Gly Ala Phe Val Asp Lys Asp Gly Thr Tyr Asp Lys Val Tyr Ile 20 25 30 CTT TTC ACT GTT ACT ATC GGC TCA AAG AGA ATT GTT AAA ATT CCG TAT 144 Leu Phe Thr Val Thr Ile Gly Ser Lys Arg Ile Val Lys Ile Pro Tyr 35 40 45 ATA GCA CAA ATG TGC TTA AAC GAC GAA TGT GGT CCA TCA TCA TTG TCT 192 Ile Ala Gln Met Cys Leu Asn Asp Glu Cys Gly Pro Ser Ser Leu Ser 50 55 60 AGT CAT AGA TGG TCG ACG TTG CTC AAA GTC GAA TTA GAA TGT GAC ATC 240 Ser His Arg Trp Ser Thr Leu Leu Lys Val Glu Leu Glu Cys Asp Ile 65 70 75 80 GAC GGA AGA AGT TAT AGT CAA ATT AAT CAT TCT AAA ACT ATA AAA CAG 288 Asp Gly Arg Ser Tyr Ser Gln Ile Asn His Ser Lys Thr Ile Lys Gln 85 90 95 ATA ATG ATA CGA TAC TAT ATG TAT TCT TTG ATA GTC CTT TTC CAA GTC 336 Ile Met Ile Arg Tyr Tyr Met Tyr Ser Leu Ile Val Leu Phe Gln Val 100 105 110 CGC ATT ATG TAC CTA TTC TAT GAA TAC CAT TAA 369 Arg Ile Met Tyr Leu Phe Tyr Glu Tyr His 115 120 122 amino acids amino acid linear protein 66 Met Ile Tyr Leu Tyr Thr Ala Asp Asn Val Ile Pro Lys Asp Gly Leu 1 5 10 15 Gln Gly Ala Phe Val Asp Lys Asp Gly Thr Tyr Asp Lys Val Tyr Ile 20 25 30 Leu Phe Thr Val Thr Ile Gly Ser Lys Arg Ile Val Lys Ile Pro Tyr 35 40 45 Ile Ala Gln Met Cys Leu Asn Asp Glu Cys Gly Pro Ser Ser Leu Ser 50 55 60 Ser His Arg Trp Ser Thr Leu Leu Lys Val Glu Leu Glu Cys Asp Ile 65 70 75 80 Asp Gly Arg Ser Tyr Ser Gln Ile Asn His Ser Lys Thr Ile Lys Gln 85 90 95 Ile Met Ile Arg Tyr Tyr Met Tyr Ser Leu Ile Val Leu Phe Gln Val 100 105 110 Arg Ile Met Tyr Leu Phe Tyr Glu Tyr His 115 120 6 amino acids amino acid single linear peptide 67 Asp Cys Gln Asn Tyr Ile 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ68 /note= “Xaa denotes N or G at residue #4; and A or S at residue #5” 68 Cys Gly Thr Xaa Xaa Xaa Xaa Pro 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ69 /note= “Xaa denotes S or C at residue #3” 69 Gly Xaa Xaa Pro Tyr Asp Pro 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ70 /note= “Xaa denotes V, N or A” 70 Leu Tyr Ser Gly Thr Xaa Ala 1 5 7 amino acids amino acid single linear peptide 71 Leu Asn Ala Pro Asn Phe Val 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ72 /note= “Xaa denotes V or I” 72 Arg Xaa Ala Arg Val Cys Lys 9 amino acids amino acid single linear peptide Peptide 1..9 /label= SEQ73 /note= “Xaa denotes T or A at residue #2; T or S at residue #3; F or Y at residue #4; and A or S at residue #7” 73 Trp Xaa Xaa Xaa Leu Lys Xaa Arg Leu 1 5 9 amino acids amino acid single linear peptide Peptide 1..9 /label= SEQ74 /note= “Xaa denotes N or D” 74 Pro Phe Tyr Phe Xaa Glu Ile Gln Ser 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ75 /note= “Xaa denotes F or Y at residue #7” 75 Gly Ser Ala Val Cys Xaa Xaa 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ76 /note= “Xaa denotes P or A at residue #6” 76 Asn Ser Asn Trp Leu Xaa Val 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ77 /note= “Xaa denotes E or D at residue #2; T, Q or S at residue #7” 77 Pro Xaa Pro Arg Pro Gly Xaa Cys 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ78 /note= “Xaa denotes A or G” 78 Asp Pro Tyr Cys Xaa Trp Asp 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ79 /note= “Xaa denotes N or G at residue #4; A or S at residue #5; Y, F, H or G at residue #6; and K, R, H, N or Q at residue #7” 79 Cys Gly Thr Xaa Xaa Xaa Xaa 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ80 /note= “Xaa denotes N or G at residue #4; A, S or N at residue #5; Y, F or H at residue #6; and K, R, H, N or Q at residue #7” 80 Cys Gly Thr Xaa Xaa Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ81 /note= “Xaa denotes N or G at residue #4; and A or S at residue #5” 81 Cys Gly Thr Xaa Xaa Xaa Xaa Pro 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ82 /note= “Xaa denotes K, F or Y at residue #2; F or Y at residue #4; F, Y, I or L at residue #5; F, Y or I at residue #6; and F or Y at residue #7” 82 Asp Xaa Val Xaa Xaa Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ83 /note= “Xaa denotes V or I at residue #1; F or Y at residue #2; F, Y or L at residue #3; F, Y, I or L at residue #4; R or T at residue #6; and T or N at residue #8” 83 Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ84 /note= “Xaa denotes V or I at residue #1; F or Y at residue #2; F, Y, I or L at residue #3; F, Y or I at residue #4; R or T at residue #6; and T or N at residue #8” 84 Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ85 /note= “Xaa denotes V or I at residue #1; F or Y at residue #2; F, Y, I or L at residue #3; F, Y, I or L at residue #4; and T or N at residue #8” 85 Xaa Xaa Xaa Xaa Phe Arg Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ86 /note= “Xaa denotes V or I at residue #1; F or Y at residue #2; F, Y or L at residue #3; F, Y, I or L at residue #4; F or Y at residue #5, R or T at residue #6, E, D or V at residue #7; and T or N at residue #8” 86 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ87 /note= “Xaa denotes R, K or N at residue #1; T or A at residue #3; T, A or S at residue #4; F, Y or L at residue #5; and K or R at residue #7” 87 Xaa Trp xaa Xaa Xaa Leu Xaa 1 5 9 amino acids amino acid single linear peptide Peptide 1..9 /label= SEQ88 /note= “Xaa denotes T or A at residue #2; T, A or S at residue #3; F, Y or L at residue #4; A, S, V, I or L at residue #7” 88 Trp Xaa Xaa Xaa Leu Lys Xaa Xaa Leu 1 5 9 amino acids amino acid single linear peptide Peptide 1..9 /label= SEQ89 /note= “Xaa denotes T, A or S at residue #2; T, A or S at residue #3; F, Y or L at residue #4; A, S, I or L at residue #7” 89 Trp Xaa Xaa Xaa Leu Lys Xaa Xaa Leu 1 5 11 amino acids amino acid single linear peptide Peptide 1..11 /label= SEQ90 /note= “Xaa denotes T or A at residue #2; and T, A or S at residue #3” 90 Trp Xaa Xaa Xaa Leu Lys Xaa Xaa Leu Xaa Cys 1 5 10 9 amino acids amino acid single linear peptide Peptide 1..9 /label= SEQ91 /note= “Xaa denotes V, L or I at residue #1” 91 Xaa Pro Xaa Pro Arg Pro Gly Xaa Cys 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ92 /note= “Xaa denotes K or Y at residue #2; F or Y at residue #4; F, Y or L at residue #5; F, Y, I or L at residue #6; and F or Y at residue #7” 92 Asp Xaa Val Xaa Xaa Xaa Xaa 1 5 7 amino acids amino acid single linear peptide Peptide 1..7 /label= SEQ93 /note= “Xaa denotes K or Y at residue #2; F or Y at residue #4; F, Y, I or L at residue #5; F, Y or I at residue #6; and F or Y at residue #7” 93 Asp Xaa Val Xaa Xaa Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ94 /note= “Xaa denotes V or I at residue #1; F, Y or L at residue #3; F, Y, I or L at residue #4; R or T at residue #6; and T or N at residue #8” 94 Xaa Tyr Xaa Xaa Phe Xaa Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ95 /note= “Xaa denotes V or I at residue #1; F, Y, I or L at residue #3; F, Y or I at residue #4; R or T at residue #6; and T or N at residue #8” 95 Xaa Tyr Xaa Xaa Phe Xaa Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ96 /note= “Xaa denotes V or I at residue #1; F, Y, I or L at residue #3; F, Y, I or L at residue #4; and T or N at residue #8” 96 Xaa Tyr Xaa Xaa Phe Arg Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ97 /note= “Xaa denotes F or Y at residue #2; F, Y or L at residue #3; F, Y, I or L at residue #4; F or Y at residue #5; R or T at residue #6; E, D, or V at residue #7; and T or N at residue #8” 97 Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ98 /note= “Xaa denotes F or Y at residue #2; F, Y, I or L at residue #3; F, Y or I at residue #4; F or Y at residue #5; R or T at residue #6; E, D, or V at residue #7; and T or N at residue #8” 98 Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ99 /note= “Xaa denotes F or Y at residue #2; F, Y, I or L at residue #3; F, Y, I or L at residue #4; F or Y at residue #5; E, D, or V at residue #7; and T or N at residue #8” 99 Val Xaa Xaa Xaa Xaa Arg Xaa Xaa 1 5 8 amino acids amino acid single linear peptide Peptide 1..8 /label= SEQ100 /note= “Xaa denotes F or Y at residue #2; F, Y, I or L at residue #3; F, Y, I or L at residue #4; F or Y at residue #5; R or T at residue #6; E, D, or V at residue #7; and T or N at residue #8” 100 Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 

What is claimed is:
 1. An isolated antibody that specifically binds human semaphorin III which comprises SEQ ID NO:54. 